Human adult stem cells from diverse origins: An overview from multiparametric immunophenotyping to clinical applications

Authors

  • Bruna R. Sousa,

    1. Department of Biochemistry and Immunology, Cell Signaling and Nanobiotechnology Laboratory, Federal University of Minas Gerais, Belo Horizonte, MG, Brazil
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  • Ricardo C. Parreira,

    1. Department of Biochemistry and Immunology, Cell Signaling and Nanobiotechnology Laboratory, Federal University of Minas Gerais, Belo Horizonte, MG, Brazil
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  • Emerson A Fonseca,

    1. Department of Biochemistry and Immunology, Cell Signaling and Nanobiotechnology Laboratory, Federal University of Minas Gerais, Belo Horizonte, MG, Brazil
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  • Maria J. Amaya,

    1. Department of Internal Medicine, Section of Digestive Diseases, Yale University School of Medicine, New Haven, Connecticut
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  • Fernanda M. P. Tonelli,

    1. Department of Biochemistry and Immunology, Cell Signaling and Nanobiotechnology Laboratory, Federal University of Minas Gerais, Belo Horizonte, MG, Brazil
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  • Samyra M. S. N. Lacerda,

    1. Department of Biochemistry and Immunology, Cell Signaling and Nanobiotechnology Laboratory, Federal University of Minas Gerais, Belo Horizonte, MG, Brazil
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  • Pritesh Lalwani,

    1. Faculdade de Ciências Farmacêuticas, Universidade Federal do Amazonas, Manaus, AM, Brazil
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  • Anderson K. Santos,

    1. Department of Biochemistry and Immunology, Cell Signaling and Nanobiotechnology Laboratory, Federal University of Minas Gerais, Belo Horizonte, MG, Brazil
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  • Katia N. Gomes,

    1. Department of Biochemistry and Immunology, Cell Signaling and Nanobiotechnology Laboratory, Federal University of Minas Gerais, Belo Horizonte, MG, Brazil
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  • Henning Ulrich,

    1. Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo, São Paulo, SP, Brazil
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  • Alexandre H. Kihara,

    1. Núcleo de Cognição e Sistemas Complexos, Centro de Matemática, Computação e Cognição, Universidade Federal do ABC, Santo André, SP, Brazil
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  • Rodrigo R. Resende

    Corresponding author
    1. Department of Biochemistry and Immunology, Cell Signaling and Nanobiotechnology Laboratory, Federal University of Minas Gerais, Belo Horizonte, MG, Brazil
    • Correspondence to: Rodrigo R. Resende, Cell Signaling and Nanobiotechnology Laboratory, Department of Biochemistry and Immunology, Bloco N4 112 and G3 86, Federal University of Minas Gerais, Av Antônio Carlos, 6627/Postal code: 30370-920, Belo Horizonte, Brazil. E-mail: resende@icb.ufmg.br or rrresende@hotmail.com

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Abstract

Stem cells are known for their capacity to self-renew and differentiate into at least one specialized cell type. Mesenchymal stem cells (MSCs) were isolated initially from bone marrow but are now known to exist in all vascularized organ or tissue in adults. MSCs are particularly relevant for therapy due to their simplicity of isolation and cultivation. The International Society for Cellular Therapy (ISCT) has proposed a set of standards to define hMSCs for laboratory investigations and preclinical studies: adherence to plastic in standard culture conditions; in vitro differentiation into osteoblasts, adipocytes, and chondroblasts; specific surface antigen expression in which ≥95% of the cells express the antigens recognized by CD105, CD73, and CD90, with the same cells lacking (≤2% positive) the antigens CD45, CD34, CD14 or CD11b, CD79a or CD19, and HLA-DR. In this review we will take an historical overview of how umbilical cord blood, bone marrow, adipose-derived, placental and amniotic fluid, and menstrual blood stem cells, the major sources of human MSC, can be obtained, identified and how they are being used in clinical trials to cure and treat a very broad range of conditions, including heart, hepatic, and neurodegenerative diseases. An overview of protocols for differentiation into hepatocytes, cardiomyocytes, neuronal, adipose, chondrocytes, and osteoblast cells are highlighted. We also discuss a new source of stem cells, induced pluripotent stem cells (iPS cells) and some pathways, which are common to MSCs in maintaining their pluripotent state. © 2013 International Society for Advancement of Cytometry

Introduction

Stem cell research is one of the most interesting fields of investigation, which has led to a better understanding of cell development and discovery of alternative treatment regimes against several diseases. In 1961, Till and McCullough documented the first properties of stem cells; self-renewal, self-generation, multipotency, and differentiation [1].

Stem cell research has undergone considerable expansion since its first isolation from mouse embryo in 1981 [2], however, the first report with human embryo-derived stem cells was published only in 1998 [3]. After the ethical conflicts that this topic raised, stem cell research has gained a new dimension with the discovery of adult stem cells [4]. In 1999, Pittenger et al. demonstrated the ability of bone marrow-derived human adult Mesenchymal Stem Cells (BM-MSC) to differentiate in diverse cell types, in vitro, revealing the multipotency of adult stem cells [5]. Adult Mesenchymal Stem Cells (MSC) are found virtually in all tissues [6] and play an important role in maintaining homeostasis and repair in case of injury during disease, through the renovation of cell repertoire. Unlike adult stem cells, embryonic stem cells can proliferate/renew indefinitely due to their ability to maintain their telomeres intact similar to what takes place in tumor cells, but in contrast to normal cells where telomerase is shortened during successive cell divisions, [7-9]. However, MSC are today's promise to regenerative medicine, due to their easy culture in vitro, their high proliferation rates, and their versatility of differentiation in many cell types, including the well-established osteoblasts, chondrocytes and adipocytes [5], as well as hepatocytes [10], neurons [11-13], and glial cells [14].

The bone marrow cell population is heterogeneous and only a minority of them is multipotent. Moreover, it is thought that an even smaller fraction is totipotent [15]. The bone marrow contains at least two main cell types: hematopoietic stem cells (HSCs), which differentiate into white and red blood cells, and mesenchymal stem cells (MSCs), which at least in vitro differentiate into osteoblast, osteoclasts, adipocytes, and chondrocytes. MSC therapy is a promising alternative for treatment of neurodegenerative diseases, some types of cancer, and tissue repair. In this article, we provide an in-depth review of the antigenic properties of these cells, their culture conditions as well as their clinical application, providing an update to a recently published review in Cytometry Part A [16].

Hematopoietic-, Bone Marrow-, and Umbilical Cord Blood-Derived Stem Cells

Hematopoietic Stem Cells (HSC)

For more than 50 years, it has been known that the bone marrow is a rich source of hematopoietic stem cells (HSCs), which are responsible for the generation and maintenance of blood cells [17]. Till and McCullough in 1961 depleted the hematopoietic cells in mice, by subjecting them to high radiation doses. Subsequently, the animals received BM cells and had their hematopoietic system restored [1]. This experiment demonstrated that BM cells are responsible for the production of blood cells, although some other contradictory reports questioned this model by the fact that the high radiation doses stressed the mice and made them an extreme damage model that was used to draw conclusions about BM cell's function [18]. They further argued that this could be due to the mature progenitor cells sustaining hematopoiesis in normal physiological conditions and hematopoietic stem cells been activated just on stress conditions [18]. But, what are these HSCs? These cells are multipotent with self-renewable capacity for all specialized blood cell types. Hematopoietic lineage phenotypes have been well characterized, principally based on LKS selection (Lin c-Kit+ Sca1+). LKS-hematopoietic stem and progenitors cells are found in the circulating blood, and in the bone marrow [19] as well as in the fetal liver [20]. Such cells are responsible for the maintenance of blood cells, and have high rate of regeneration. Every day, billions of new blood cells are produced [21]. It is thought that 1 in 10,000 to 15,000 BM cells is a stem cell; in the blood this number changes to 1 in 100,000 [19]. Studies have shown that HSCs reside in the endosteal region [22, 23] and near blood vessels in the bone marrow [24]. Such regions are formed by osteoblasts, Cxcl12 expressing abundant-reticular cells, nestin-positive mesenchymal stem cells, Schwann cells, and perivascular cells [25-29]. Nilsson et al. showed that osteopontin, an overexpressed protein in osteoblasts, is a key molecule in the maintenance, regulation, proliferation, and physical localization of HSC in the BM stem cell niche [30, 31]. At least three cell populations compose the BM: long-term (LT)-HSC, short-term (ST)-HSC, and multipotent progenitor cells [32]. BM stem cells continuously go from the bone marrow to the blood circulation and return back to bone marrow, this way the circulating blood is a source of MSC and the bone marrow transplant can be used for the treatment of blood diseases [33]. Moreover, stem cell transplant is a good approach for treating several pathological conditions. Hundreds of leukemia patients were treated with BM transplant in the 1970s, in the pioneering studies by Thomas et al. [34] and many other diseases were also treated with such approach (for review see Ref. [35]). Recent work suggests that the success of engraftment depend on exogenous factors, such as, the presence of immune-regulatory T cells that can lead to failure of allogeneic bone marrow grafts [36]. Nevertheless, there is no doubt about the therapeutic importance of HSC and their differentiation capacity. Moreover, recent studies demonstrated that multipotent cells of hematopoietic lineage can also differentiate in hepatocytes, which further increases their therapeutic potential [37, 38].

Bone Marrow-Derived Stem Cells

The bone marrow is a source not only of hematopoietic stem cells, but also for mesenchymal stem cells or mesenchymal stromal cells (MSCs) or bone marrow stem cell (BM-MSC) or bone marrow stromal cells (BMSCs) [39-42]. The first report on BM-MSC was published by Friedenstein et al. in 1966, in which these cells were isolated from mouse bone marrow [39], and their fibroblastic form was observed [43]. Their work in 1974 was decisive for the establishment of the so-called fibroblast-like cells due to the resemblance to BM-MSCs [43-47]. Human MSCs (hMSCs) were isolated by Pittenger et al. in 1999 from the pelvic iliac crest [5]. In 2002, Murphy et al. and D' Ippolito et al. isolated hMSCs from the tibia and the femoral region, respectively [48-50]. The HSCs correspond to a small fraction of the adherent cells in vitro, these cultured BM-MSCs are primarily adherent and by definition, able to differentiate in osteoblasts, chondroblasts, and adipocytes [51]. However, several studies have shown the ability of BM-MSCs to differentiate not only in cell types that originated from the mesoderm, but also from the ectoderm [12, 47, 52, 53]. Researchers have obtained glial, muscle and hepatic cells from BM-MSC [37, 54, 55]. Sanchez-Ramos et al. differentiated mouse BM-MSCs into immature neurons [13]. Being very heterogeneous, the bone marrow cell population forms the niche, or “stroma” responsible for the maintenance of HSCs, promoting survival and adhesion factors for them [56]. Such “niche” is formed by the Stromal Cells (reticular cells, fibroblasts, osteoprogenitors, primary cellular components of the marrow stroma) [57]. The BM-MSC culture is composed of colony-forming units-fibroblastic (CFU-F)—a mix of tri-, bi-, and unipotent cells. The relative rate of these populations determines the potential of their growth, senescence and differentiation (in a multipotent manner) in the BM-MSC culture. Because of the heterogeneity of these cell populations, BM-MSCs possess distinct differentiation potentials. Muraglia et al. obtained 30% of “Tri-lineage,” the rest in “bi-lineage” and “uni-lineage” in a BM-MSC culture [58]. This is likely due to the fact that BM-MSC has subpopulations of MSC in different stages of differentiation and the homogeneity of the population is dependent on the tissue of origin, isolation method and passage number. However, a myriad of studies show the capacity of MSC to differentiate in adipose [44], tendon [59], muscle [55, 60], cartilage [61], bone [62], and nervous tissues [12, 13, 53]. Experimental evidence also supports the stem cell character of pericytes and their differentiation potential [63]. This affirmation is in agreement with a recent flow cytometry study suggesting the universal character of pericytes as stem cells and perivascular origin of MSC [64, 65].

Umbilical Cord Blood-Derived Stem Cells

Another important source of MSC is the umbilical cord blood (UCB-MSC) [66]. It is already a routine procedure to cryopreserve the UCB with the intention to use them as an alternative for future treatments in autologous transplants. Although opposing arguments [67] show that the cell population of the UCB, as well as the BM, harbor pluripotent/multipotent stromal cells, which are able to differentiate, for instance, in neurons with 87% success rate [68], as well as osteoblasts, chondroblasts, and adipocytes. Goodwin demonstrated the multilineage potential of (adherent) cells isolated from the umbilical cord blood, which expressed neuronal, bone and adipose tissue markers. However, the authors were very cautious not to approach what can be “cellular plasticity” as “pluripotency” [69] and referred these cells as “non-progenitor” (NHP) instead of stem cells. This same caution should be attributed to BM-MSCs, since they share surface markers such as the neuronal proteins GFAP (Glial fibrillary acidic protein) and TuJ1 (β-tubulin) [70] among others (for review see [71]), with specialized cell types (Table 1). Adherent cells isolated from the UCB (UCB-MSC) consist of two populations: osteoclast and mesenchymal-like phenotype, according to Erices et al. At least 75% have the morphology and the characteristics of multinucleated osteoclasts. On the other hand, 25% initially originated from individual fibroblastoid colonies with high proliferation rates [72]. In addition to the blood, the cord stromal cells have been explored as a source of MSC. UC-MSCs show cardiomyocyte like-phenotype [73]. UCB-MSCs also have an interesting immunoregulatory property. They suppress lymphocyte proliferation and decrease pro-inflammatory cytokine levels (interferon-γ, tumor necrosis factor-α, β (TNF-α, TNF-β). In fact, models of cerebral ischemia, hepatic cirrhosis and pulmonary fibrosis have shown significant improvement and inflammation control, as well as reduction of collagen resulting in an anti-fibrotic effect, days after UC-MSC infusion (for review see Ref. [74]).

Table 1. Well-known cell surface markers
Cell type (human)MarkerReferences
  1. CD = Cluster of differentiation; HLA = human leukocyte antigen; SH3 = Src homology 3 domain; SH2 = Src homology 2 domain; Thy-1 = thymocyte antigen; STRO-1 = stromal precursor antigen-1; VCAM-1 = vascular cell adhesion molecule-1.

Umbilical cord mesenchymal stem cells (UC-MSC)CD29, CD44, CD49b, CD105 (SH2), CD166, HLA-ABC[72, 73]
Bone marrow mesenchymal stem cells (BM-MSC)CD29, CD44, CD73 (SH3), CD90 (Thy-1), Stro-1, CD106 (VCAM-1) CD105, CD166, HLA-ABC,[261-263]
Hematopoietic mesenchymal stem cells (HSC)CD34, CD38, CD59, CD133[264]
OsteoblastCD45, CD51/CD61[73]
NeuronGrowth Associated Protein-43 (GAP-43), neurofilament-H (NF-H), Neuronal Nuclei (NueN), Class-III β-tubulin (TuJ-1)[265]

Surface Markers of Hematopoietic-, Bone Marrow-, and Umbilical Cord-Derived MSCs

In 2006, the Mesenchymal and Tissue Stem Cell Committee of the International Society for Cellular Therapy defined the minimal criteria that MSC should present: to be adherent in culture conditions, to express some surface markers such as CD105, CD73 and CD90, not to express CD45, CD34, CD14, CD11b, CD79a or CD19 and HLA-DR [42]. Nevertheless, some of these markers are not exclusive for MSC [71, 75, 76]. The different tissues origin may be related to the differences in their expression. However, in all cases, the general rule of positive markers is CD73, CD90, and CD105 and the respective negatives as shown above, as a minimal criterion of expression for MSC in therapy. There is a wide variety of isolation, culture and induction protocols for blood, bone marrow and umbilical cord-derived MSCs summarized in Tables 2 to 4 (Fig. 1). Recently published manuscripts, summarize flow cytometry characterization and specification of these stem cell based on phenotypic and differentiation stage-specific markers, for isolation and studying their biology and clinical applications [16, 20, 64, 77, 78]. Multiparametric flow cytometry and image stream analysis, the latter one combining morphological and imaging analysis with population-relevant antigene expression are being used for identifying tissue-specific stem cells, as well as for tumor cells and subpopulations within them in humans and animals [79-83].

Table 2. Isolation, characterization and differentiation of human adipose-derived stem cells (hASCs)
Isolation protocolCharacterizationDifferentiation protocolReferences
  1. K-NAC medium = N-acetyl-l-cysteine (NAC) and L-ascorbic acid 2-phosphate; FCS = fetal calf serum; HLA = human leukocyte antigen; CD = cluster of differentiation; STRO-1 = stromal precursor antigen-1; PBS = phosphate buffered saline; EDTA = ethylenediaminetetraacetic acid; DMEM/F12 = Dulbecco's modified eagle medium/nutrient mixture F-12; ITS = insulin-transferrin-selenium; TGF = transforming growth factor; IGF = Insulin-like growth factor; α-MEM = α-modified Eagle's medium; . hADSC = human adipose stem cell; DMEM-LG = Dulbecco's modified Eagle's medium, low glucose; FBS = fetal bovine serum; rhEGF = recombinant human epidermal growth factor; MCDB-201 = molecular, cellular, and developmental biology medium; FN = fibronectin; rhOSM = recombinant human oncostatin M; rhHGF = recombinant human growth factor; MEM = Modified Eagle's medium; FGF = fibroblast growth factor; SCF = stem cell factor; ABCG2 = ATP-binding cassette subfamily G member 2; Thy-1 = thymocyte antigen; ISL-1 = transcription factor islet-1; Human A-MSC = human adipose-derived mesenchymal stem cells; DMEM = Dulbecco's modified Eagle's medium; NH4Cl = ammonium chloride; ITM = inorganic trace minerals; MHC = major histocompatibility complex; hEGF = human epidermal growth factor; BDNF = Brain-derived neurotrophic factor; RA = retinoic acid; ASCs = adipose-derived stem cells; VEGF = vascular endothelial growth factor; EGM-2-MV = microvascular endothelial cell growth medium-2; SSEA4 = stage-specific embryonic antigen-4; IBMX = isobutyl-methylxanthine.

ASCs from lipoaspirate were washed with PBS. The sample was digested with collagenase. K-NAC medium was added to the cell pellet. Cell suspension was remixed, filtered, plated and incubated.Positive cellular markers: CD106, CD13, CD49, CD44, CD90, CD105, CD29, and CD166. Negative cellular markers: HLA-DR, CD133, CD117, CD45, CD31, STRO-1, HLA-II and CD34.Osteogenic: ascorbate-2-phosphate, β-glycerolphosphate and dexamethasone.[266]
The lipoaspirate was washed with PBS. The cells were seeded in culture flasks. After passage 2, the cells were suspended in cryopreservation medium.Positive cellular markers: CD90, CD73, and. CD166. Negative cellular markers or low levels: CD49, CD45, and CD34.Chondrogenic: DMEM/F-12, ascorbate-2-phosphate, l-proline, dexamethasone, sodium pyruvate, ITS, single or combinations of TGF-β2, TGF-β2 and IGF-I.[267]
ASCs from abdominoplasty were washed with PBS, centrifuged and resuspended in medium α-MEM. After the cells were suspended in expansion medium.Positive cellular markers: CD29, CD90, CD105 and CD44. Negative cellular markers: HLA-DR, CD14, CD45, and CD34.Hepatic: expansion medium without serum, rhOSM dimethyl sulfoxide and rhHGF.[268]
ASCs from lipoaspirate were suspended in DMEM. Cells were collected and washed with DMEM/F12 medium.Positive cellular markers: c-kit and stem cell factor (SCF). Negative cellular markers: ABCG2, nestin, Thy-1, and Isl-1.Pancreatic: DMEM/F12 medium with glucose, penicillin/streptomycin, nicotinamide, hepatocyte growth factor, pentagastrin, B-27activin-A, exendin-4, serum-free supplement and N-2 Supplement.[269]
hASC from lipoaspirate were suspended in DMEM and centrifuged. The pellet was resuspended in NH4Cl and filtered. The cells were cultured in DMEM.Positive cellular markers: MHC I, CD90, CD73, CD44, CD29 and CD105 (endoglin). Negative cellular markers: CD34, CD45, CD31 and CD14.Neurogenic: serum-free medium, hEGF, basic FGF, poly-l-lysinated coverslips, BDNF, FBS and all-trans RA.[270]
ASCs from lipoaspirate were washed with PBS, suspended in DMEM and centrifuged. The pellet was resuspended in NH4Cl and incubated in DMEM medium.Positive cellular markers: CD105, CD90 and CD73. Negative cellular markers: CD34 and CD45.Cardiac: DMEM-LG, insulin, transferrin, sodium selenite, FCS, antibiotics bovine serum albumin, ascorbate, dexamethasone and linoleic acid.[271]
hASCs were isolated from lipoaspirate, washed with PBS, digested with collagenase, filtered and centrifuged. The hASCs were cultured in standard incubation conditions.Positive cellular markers: CD9, CD29, CD49, CD54, CD105, CD166, CD44, CD71, CD10, CD13, CD73, CD90 and CD146. Negative cellular markers: CD11b, CD18, C50, D56, CD62, CD104, CD16, C14, CD31, CD45 and HLA-DR.Adipogenic: Dexamethasone, isobutyl methylxanthine, indomethacin, insulin and thiazolidinedione. Cardiomyogenic: Transferrin, IL-3, IL-6 and VEGF. Chondrogenic: Ascorbic acid, bone morphogenetic protein 6, dexamethasone, insulin and transforming growth factor-β. Endothelial: Proprietary medium (EGM-2-MV; Cambrex), ascorbate, epidermal growth factor, basic fibroblast growth factor and hydrocortisone. Myogenic: Dexamethasone and horse serum. Neurogenic: Butylated hydroxyanisole, valproic acid and insulin. Osteogenic: Ascorbic acid, bone morphogenetic protein 2, dexamethasone and 1α,25-dihydroxyvitamin D3.[111]
hASCs were isolated from lipoaspirate through collagenase treatment followed by centrifugation and differential platting technique.Positive cellular markers: CD9, CD10, CD13, CD29, CD44, CD49e, CD51, CD55, CD59, CD90 and CD166. Negative cellular markers: CD11a, CD11b, CD11, CD14, CD16, CD18, CD31, CD45, CD50, CD56, CD104 and HLA-DR.Adipogenic: insulin-like growth factor-1 (IGF-1), growth hormone, glucocorticoids, insulin, fatty acids and cyclic adenosine monophosphate. Osteogenic: dexamethasone, ascorbic acid, β-glycerophosphate and growth factor bone morphogenetic protein 2. Chondrogenic: insulin, TGF-β1 and ascorbate. Myogenic: complete media, horse serum and glucocorticoid such as hydrocortisone and/or dexamethasone.[272]
hASCs were isolated from lipoaspirate, digested with collagenase. Cells obtained were separated through density gradient centrifugation, collected, washed and filtrated.Positive cellular markers: CD34 and CD90. Negative cellular markers: CD31, CD45, CD105 and CD146.Adipogenic: DMEM, isobutylmethylxanthine, dexamethasone, insulin and indomethacin. Osteogenic: DMEM, FBS, dexamethasone, ascorbate-2-phosphate and β-glycerophosphate. Chondrogenic: DMEM, FBS, insulin, TGFβ1and ascorbate-2-phosphate.[273]
Table 3. Isolation, characterization and differentiation of human umbilical cord blood mesenchymal stem cell (UCB-MSCs)
Isolation protocolCharacterizationDifferentiation protocolReferences
  1. EBSS = Earle's balanced salt solution; DMEM-LG = Dulbecco's modified Eagle's medium, low glucose; HEPES = N−2-hydroxyethylpiperazine-N-ethanesulfonic acid; FBS = fetal bovine serum; UC = umbilical cord; RPMI1640 = Roswell Park Memorial Institute medium; CD = cluster of differentiation; SH3 = Src homology 3 domain; ICAM-1 = intercellular adhesion molecules-1; Thy-1 = Thymocyte antigen; ASMA = Alpha-smooth muscle actin; SH2 = Src homology 2 domain; IMDM medium = Iscove's modified Dulbecco's medium; HGF = human growth factor; FGF = fibroblast growth factor; ITS = Insulin-transferrin-selenium; DMEM = Dulbecco's modified Eagle's medium; BSA = bovine serum albumin; BME = β-mercaptoethanol; cAMP = cyclic adenosine monophosphate; AsA = ascorbic acid; aFGF = acidic Fibroblast Growth Factor; SHH = Sonic hedgehog; BDNF = Brain-derived neurotrophic factor; NGF = nerve growth factor; IBMX = isobutyl-methylxanthine; PMA = phorbol myristate acetate.

The umbilical cord was washed with EBSS and filled with collagenase in 199 medium. The sample was centrifuged and the pellet was resuspended in DMEM-LG. Cells were cultured and after 4 days cells were passaged and transferred to a flask for expansion.Positive cellular markers: CD73/SH3, CD54 (ICAM-1), CD29 (β1 integrin), CD49e (α5 integrin), CD44, CD90 (Thy-1), ASMA, CD105/SH2/endoglin and CD13. Negative cellular markers: CD14, CD45 and CD34.Adipogenic: isobutyl-methylxantine, dexamethasone, insulin and indomethacin[274]
Osteogenic: dexamethasone, β-glycerophosphate and ascorbate-phosphate[274]
Hepatic: Step-1: IMDM, HGF, bFGF and nicotinamide; Step-2: IMDM, oncostatin M, dexamethasone and ITS+ premix[10]
Chondrogenic: DMEM high-glucose, dexamethasone, ascorbic acid, sodium pyruvate, proline, transforming growth factor-β, ITS+ (insulin, transferrin, selenium), BSA and linoleic acid[10]
  Cardiac: culture medium, horse serum, dexamethasone and hydrocortisone.[275]
Neurogenic: Step1: medium consists of IMDM, bFGF, retinoic acid and BME. Step 2: IMDM 1, cAMP and AsA. Step 3: IMDM, hydrocortisone and cAMP. Step 4: IMDM, aFGF, SHH, BDNF, NGF, vitronectin, AsA, IBMX, forskolin and PMA.[276]
Table 4. Isolation, characterization and differentiation of human Mesenchymal Stem Cell and human Hematopoietic Stem Cell (hHSC)
Isolation protocolCharacterizationDifferentiation protocolReferences
  1. AsA = ascorbic acid; Asc P = ascorbic acid 2-phosphate; βGP = β-glycerophosphate; FCS = fetal calf serum; HLA = human leukocyte antigen; CD = cluster of differentiation; 1; PBS = Phosphate Buffered Saline; EDTA = ethylenediaminetetraacetic acid; DMEM = Dulbecco's Modified Eagle medium; MSCGM = mesenchymal stem cell growth medium; MSCGS = mesenchymal stem cell growth supplement; IMDM = Iscove's Modified Dulbecco's media; HGF = human growth factor, ITS = Insulin-transferrin-selenium; IBMX = 3-isobutyl-1-methyl-xanthine; TGF = transforming growth factor; BME = β-mercaptoethanol; DMSO = dimethyl sulfoxide; BHA = butylated hydroxyanisole; BSA = bovine serum albumin; cAMP = cyclic adenosine monophosphate, FBS = fetal bovine serum; FCS = Fetal calfserum; EGF = HUMAN, aFGF = acidic fibroblast growth factor, bFGF = basic fibroblast growth factor; SHH = Sonic hedgehog; epidermal growth factor; HGF = human growth factor; NGF = nerve growth factor; PMA = phorbol myristate acetate; Thy-1 = Thymocyte antigen; EGF = Epidermal Growth Factor; BDNF = brain-derived neurotrophic factor; IBMX = isobutyl-methylxanthine; IL-3 = Interleukin 3; RPMI = Roswell Park Memorial Institute; VEGF = vascular endothelial growth factor.

Human mesenchymal stem cell   
BM was aspirated and diluted with EDTA-PBS. The MNC fraction was isolated by density gradient centrifugation and seeded at a density of 1 × 106 cells per cm2Positive cellular markers: CD44, CD71, CD73 (SH3), CD90 (Thy-1), CD105, CD29, HLA I, CD106 (VCAM-1) and CD166.Osteogenic: dexamethasone, AsA, AsAP, βGP, and FCS in osteogenic basal medium[277]
 Negative cellular markers: CD10, CD13, CD14, CD24, CD31, CD34, CD36, CD45, CD38, CD45, CD49d, CD117, CD133, SSEA4 and HLA-DR.Adipogenic: Adipogenic Differentiation: MSCGM or DMEM, MSCGS, dexamethasone, IBMX, recombinant human insulin, indomethacin and FCS[278]
  Chondrogenic: DMEM high-glucose, dexamethasone, ascorbic acid, sodium pyruvate, proline, TGF-β1, ITS+ (insulin, transferrin, selenium) premix[10]
Human bone marrow was aspirated, separated by negative immunodepletion followed by density-gradient centrifugation and plated in tissue culture flasks in expansion mediumPositive cellular markers: CD29, CD44, CD73, CD90 and CD105Hepatic: IMDM medium serum free, epidermal growth factor and bFGF. Step 1: IMDM, HGF, bFGF and nicotinamide. Step 2: IMDM, oncostatin M, dexamethasone and ITS+ premix[10]
 Negative cellular markers: CD13, CD34, CD45 and CD133Neurogenic: DMEM, FBS, β-mercaptoethanol. Neuronal induction media composed of DMEM/BME. In later experiments DMEM/dimethylsulfoxide (DMSO) and BHA276
  Osteogenic: dexamethasone, ascorbic acid and β-glycerophosphate[279]
  Chondrogenic: DMEM serum-free, ascorbic acid-2-phosphate, sodium pyruvate, proline, dexamethasone, ITS + premix, TGF-β3 and bFGF[280]
Human peripheral blood is separated by Ficoll-Paque. The cells were extracted, transferred into tube and centrifuged. After the cells were washed with culture media and centrifuged. Cell pellet was resuspended and plated in culture media 10 to 20% FBSPositive cellular markers: CD29, CD71, CD38, CD90 (Thy-1), 105, CD166.Adipogenic: IBMX, dexamethasone, insulin, indomethacin[279]
 Negative cellular markers: CD34, CD45 and CD38.Red Blood Cells: deionized BSA, iron-saturated human transferrin, ferrous sulfate, ferric nitrate, insulin, hydrocortisone, stem cell factor, IL-3 and erythropoietin.[281]
  Macrophage: RPMI medium 1640, FBS, l-glutamine, M-CSF-treated monocyte cultures and LPS. T Lymphocyte: RPMI medium 1640, FBS, l-glutamine and IL-2. Epithelial: RPMI medium 1640, FBS, l-glutamine and EGF. Endothelial: RPMI medium 1640, FBS, l-glutamine and VEGF. Hepatic: RPMI medium 1640, FBS and HGF. Neurogenic: RPMI medium 1640, FBS, l-glutamine and NGF[282]
Table 5. Isolation, characterization and differentiation of menstrual blood-derived mesenchymal stem cells
Isolation protocolCharacterizationDifferentiation protocolReferences
  1. FBS = fetal bovine serum; CD = cluster of differentiation; hTERT = human telomerase reverse transcriptase; Oct-4 = octamer-binding transcription factor 4; STRO-1 = stromal precursor antigen-1; SSEA-4 = stage-specific embryonic antigen-4; NPMM = neural progenitor maintenance medium; hFGA = human fibroblast growth factor; SAGM induction medium = small airway growth medium; FGF = fibroblast growth factor; hFGF = Human fibroblast growth factor; SCF = stem cell factor; DMEM = Dulbecco's modified Eagle's medium; DMEM/F12 = Dulbecco's modified eagle medium/nutrient mixture F-12; PBS = Phosphate Buffered Saline; ITS = insulin-transferrin-selenium; EGF = epidermal growth factor; HGF = hepatocyte growth factor; OSM = oncostatin M; NTA = nitrilotriacetic acid trisodium.

Mononuclear cells derived from menstrual blood were separated through density gradient centrifugation and pre-cultured in common medium. Subsequently, cells were collected and subcultured.Positive cellular markers: CD9, CD29, CD59, CD73, CD41a, CD44, CD90, CD105, hTERT and Oct-4. Negative cellular markers: CD14, CD34, CD38, CD45, CD133, STRO-1, SSEA-4 and Nanog.Adipogenic: Commercial Adipogenic Induction media. Osteogenic: Commercial Osteogenic Induction media. Endothelial: Commercial Endothelial Induction media. Neurogenic: Commercial NPMM neural induction media, penicillin/streptomycin, glutamax and hFGA-4. Pulmonary epithelial: Commercial SAGM induction medium. Hepatic/pancreatic: Commercial induction medium, hepatocyte growth factor, FGFβ, hFGF-4 and SCF. Cardiogenic and myogenic: complete DMEM, 5-Azacytidine, followed by cultivation with Skeletal Muscle Growth Medium and b-FGF.[119]
Cells derived from menstrual blood were washed in complete media, seeded in culture flasks and grown in Chang's Complete Media.Positive cellular markers: SSEA-4, Oct-4, CD117low, CD29, CD44, CD166, CD73 and CD90. Negative cellular markers: CD133, CD45, CD105 and CD34.Osteogenic: Commercial induction differentiation kit. Adipogenic: Commercial induction differentiation kit. Chondrogenic: Commercial induction differentiation kit. Cardiomyogenic: Commercial induction differentiation kit. Neurogenic: Commercial induction differentiation kit.[126]
Mononuclear cells derived from menstrual blood were separated through density gradient centrifugation and washed with PBS. The cell pellet was suspended in complete DMEM-F12 and cultured.Positive cellular markers: CD105, CD73, CD146 and OCT-4. Negative cellular markers: c-kit and CD45.Osteogenic: DMEM, fetal bovine serum, dexamethasone, β-glycerophosphate and ascorbate–phosphate. Adipogenic: DMEM, FBS, dexamethasone, recombinant human insulin, isobutyl-1-methyl-xanthine and indomethacin. Chondrogenic: medium containing FBS, transforming growth factor-b3, bone morphogenetic protein 6, dexamethasone, 1 × ITS + 1 (ITS + bovine serum albumin and linoleic acid) and ascorbic acid. Hepatic: Commitment step: platting extracellular matrix gel with serum-free DMEM supplemented with EGF and bFGF. Differentiation step: serum-free medium containing dexamethasone, 1% ITS + 1, NTA and HGF. Maturation step: serum-free medium containing dexamethasone, 1% ITS + 1 and OSM.[121]
Table 6. Clinical applications: In vivo studies
Cell typeCondition or pathologySpeciesVia and type of transplantationResultsReferences
  1. ASCs = adipose derived stem cells; mdx mice = mice with X-linked dystrophy; PLGA = poly(lactic-co-glycolic acid); RAG2 = recombination activating gene 2; MyoD = myogenic differentiation factor D; hMADS = human multipotent adipose-derived stem; ACHMS = atelocollagen honeycomb-shaped scaffold; ADSCs = adipose derived stem cells; β-TCP = β-tricalcium phosphate; BMP-2 = Bone morphogenetic protein-2; hASC = human adipose stem cell; GFP = green fluorescent protein; LV = left ventricular; SSEA-1 = anti-stage-specific embryonic antigen-1; SCI = spinal cord injury; hADSCs = human adipose derived stem cells; PDX-1 = pancreatic-duodenal homeobox 1; CT = computed tomographic; SCID = severe combined immunodeficient mice; MMCs = menstrual blood-derived mesenchymal cells; MBSCs = menstrual blood stem cells.

Adipose-derived Mesenchymal Stem Cells (ASCs)Muscular dystrophyHind-limb ischemia mice model and mdx miceIntramuscular injection. Allogeneic.Incorporation of ASCs into muscle fibers and significant restoration of dystrophin expression in mdx mice[128]
 Muscle regenerationNude miceASCs were injected subcutaneously. Autologous.Newly formed muscular tissues in mice that received ASCs attached to PLGA[129]
 Muscular dystrophyRAG2−/− γC−/− micehASC were injected intramuscularly. Xenogeneic.Increase in the number of hMADS cell-derived fibers[130]
 OsteogenesisNude miceACHMS scaffold were transplanted subcutaneously. Xenogeneic.Fibrous tissue formation, vascularization, calcifications and high radio-opacities were observed[131]
 Critical-sized Skull defectRabbitScaffolds of ADCs were grafted in the skullcap. Autologous.Enhanced bone formation[132]
 Critical Calvarial defect.Nude ratOsteo-induced human ASC in PLGA scaffold were implanted in the skullcap. Xenogeneic.Bone regeneration capability in critical-sized skeletal defects[133]
 Critical-sized Segmental Radial defectRabbitASCs encapsulated in collagen I gel with PLGA-β-TCP scaffolds were implanted in radial defects. Autologous.Osteogenesis in critically sized orthotopic site[134]
 Femoral defectNude ratGenetically modified BMP-2 hASCs were implanted in femoral defect. Xenogeneic.Induction of bone formation in vivo and healing of critically sized femoral defect[135]
 Tendon InjuryRabbitTopical injection of ASCs were injected. Autologous.Significant increase in tensile strength, differentiation of ASCs toward tenocytes and endothelial cells and increases in angiogenic growth factors[137]
 Myocardial infarctionNude ratIntramyocardial human ASCs were injected. Xenogeneic.Human ASCs preserved heart function, augmented local angiogenesis and cardiac nerve sprouting[139]
 Myocardial infarctionPigThe cells were injected intracoronary. Autologous.Improvements cardiac function and perfusion via angiogenesis[144]
 Myocardial infarctionLewis ratIntramyocardial. Autologous.Improvements cardiac function of infarcted rat hearts[148]
 Myocardial infarctionLewis ratLeft ventricular (LV) chamber injection of rat GFP-ASCs. Allogeneic.LV end-diastolic dimension was less dilated, the ejection fraction and cardiac were significantly improved[143]
 Myocardial infarctionC57Bl/6 N miceGFP-ASCs intramyocardial injection. Allogeneic.The ASCs were able to promote neo-vascularization in the ischemic heart[141]
 Coronary Artery occlusionRhesus monkeysInfarction area was covered with a composite sheet harboring both ASCs and SSEA-1+ cardiac progenitors. Autologous.The ASCS increased angiogenesis[154]
 Cerebral IschemiaRatIntracerebral injection of neural induced- rat ASCs. Allogeneic.Functional recovery and more reduction in hemispheric atrophy[159]
 Sciatic Nerve injurySprague-Dawley ratASCs differentiated towards to Schwann cells were implanted in transected nerve ends. Allogeneic.A significantly enhanced nerve regeneration distance[160]
 Acute Spinal Cord injuryDogInjection into SCI site. Allogeneic.Improvements of neurological function and neural differentiation[165]
 Spinal Cord injuryRatInjection into SCI site. Allogeneic.Significant recovery from hindlimb dysfunction. Neurotrophic factors promoted functional recovery[163]
 Hindlimb ischemiaNude miceIntramuscular transplantation of human ASCs cultured as spheroids. Xenogeneic.Improvements cell survival, angiogenic factor secretion, neovascularization, and limb survival[157]
 Acute liver injuryNude miceTail vein injection of human ASC-derived hepatocyte. Xenogeneic.Incorporation into the parenchyma of the liver[167]
 Liver injury and partial hepatectomySprague-Dawley ratThe cells were injected via hepatic portal vein or the dorsal vein of the penis. Allogeneic.The cells decreased the level of serum liver enzymes such as alanine aminotransferase and aspartate aminotransferase and improved serum albumin and liver function[168]
 Diabetes mellitus type1C57BL/6J miceTransfected Pdx1-ASCs were injected by tail vein. Allogeneic.Pdx1-ASCs acquired functional beta-cell phenotype, and partially restored pancreatic function in vivo[170]
 Linear Scleroderma “en coup de sabre” deformityHumanFacial lipoinjection in combination with ASCs. Autologous.Improvement of appearance. Substantial enhanced result in regarding one session of treatment[173]
 Facial LipoatrophyHumanTopical lipoinjection in combination with ASCs. Autologous.No statistically significant difference in clinical improvement score compared to the conventional lipoinjection.[172]
 Breast reconstructionHumanTopical lipoinjection in combination with ASCs. Autologous.Improvements fat grafting with retention of volume[171]
 Treacher Collins syndromeHumanSculpted bone allograft, ASCs BMP-2 and periosteal grafts were utilized. Autologous.CT scan demonstrated complete bone reconstruction of the bilateral zygomas.[283]
 Widespread calvarial defectHumanLocal injection associated to fibrin glue. AutologousNew bone formation and near complete calvarial continuity three months after the reconstruction.[174]
 Large calvarial defectHumanImplantation of ASCs seeded in β-TCP scaffold. AutologousNo complications and satisfactory outcome in ossification[176]
 Hemi-maxillectomy due to a large keratocystHumanImplantation of titanium cage filled with ASCs, β-TCP and BMP-2. AutologousImplants osseo-integration without any adverse events[175]
 Femoral head osteonecrosisHumanLocal injection of ASCs with calcium chloride-activated platelet rich plasma and hyaluronic acid. Autologous.Regeneration of medullary bone-like tissue and long-term reduction of hip pain[284]
 Acute myocardial ischemia and left (LV) ventricular impairmentHumanThe cells were injected intracoronary. Autologous.Improvement of cardiac function, LV ejection fraction and reduction in infarct size in the ASC-treated group[285]
 End-stage Coronary Artery disease and severe LV dysfunctionHumanTransendocardial. Autologous.Ongoing study. Results not available[285]
 Persistent Sterile Corneal Epithelial defectHumanTopical injections. Autologous.Complete corneal epithelial healing was observed 1 month after transplantation[179]
 Chronic fistulas in Crohn's diseaseHumanEnterocutaneous fistula-injection into the wall of the track associated to fibrin glue. Rectovaginal and perianal fistulas- injections into the rectal mucosa. Autologous.The proportion of patients who achieved fistula healing was significantly higher with ASCs than with fibrin glue alone[180-182]
Menstrual blood-derived Mesenchymal Stem CellsDuchenne muscular dystrophymdx-scid miceThe cells were injected intramuscular. Xenogeneic.Human menstrual blood-derived cells transplantation improved the efficiency of muscle regeneration and dystrophin delivery to dystrophic muscle in mdx-scid mice[185]
 Myocardial InfarctionF344 nude ratThe cells were injected into the myocardium. Xenogeneic.Transplanted MMC-derived cardiomyocytes significantly restored impaired cardiac function, decreasing the myocardial infarction area in the recipient rat heart[186]
 Multiple Sclerosis.humanThe cells were injected intrathecal and intravenous. Allogeneic.Disease progression did not occur in the patients treated. Abnormality, immunological reactions or treatment-associated adverse effects were not evidenced[189]
 Cerebral IschemiaSprague-Dawley ratThe cells were injected intracerebral and intravenous. Xenogeneic.Transplanted MBSCs significantly reduced behavioral abnormalities, ameliorating motor and neurological impairments[188]
 Critical Limb IschemiahumanThe cells were injected intramuscular. Allogeneic.Initiating clinical studyNCT01558908
Table 7. Isolation, characterization, and differentiation of human amnion epithelial cells
Isolation protocolCharacterizationDifferentiation protocolReferences
  1. DMEM = Dulbecco's modified Eagle's medium; FBS = fetal bovine serum; HLA = human leukocyte antigen; MHC = major histocompatibility complex; DMEM-H = Dulbecco's modified Eagle's medium high; TGF = transforming growth factor; IBMX = isobutyl-methylxanthine; HBSS = Hanks' balanced salt solution; EDTA4Na = tetrasodium ethylenediaminetetraacetic acid; SSEA-4 = stage-specific embryonic antigen-4; TRA 1–81 = tumor rejection antigen 1–81; SSEA-3 = stage-specific embryonic antigen-3; TRA1–60 = tumor rejection antigen1–60; SSEA-1 = stage-specific embryonic antigen-1; FGF = fibroblast growth factor.

Treatment with dispase II, cultivation in Dulbecco's modified Eagle medium (DMEM, glutamine, high glucose, FBS, inactivated)Positive cellular markers: CD166, CD105, CD90, CD73, CD49e, CD44, HLA-ABC, CD29 and CD13. Negative cellular markers: CD49e, CD14, CD34, CD45 and MHC II.Chondrogenic: DMEM-H, fetal bovine serum (FBS), insulin, TGF-β1 and fresh ascorbic acid. Osteogenic: DMEM-H, FBS, β-glycerophosphate, 1α,25-dihydroxyvitamin D3, ascorbic acid and dexamethasone. Adipogenic; DMEM-H/FBS/dexamethasone/isobutyl-methylxanthine (IBMX)/indomethacin and insulin; Myogenic: DMEM-H/FBS/hydrocortisone/dexamethasone and horse serum; Neurogenic: DMEM-H/FBS and all trans retinoic acid[286]
The amnion layer was washed with Hanks' balanced salt solution (HBSS) and incubated with trypsin containing EDTA4Na. The cells digests were washed with HBSS.Positive cellular markers: SSEA-4, TRA 1–81, SSEA-3, and TRA1–60. Negative cellular markers: SSEA-1 and CD34Cardiomyogenic: DMEM, FBS, 2-mercaptoethanol, sodium pyruvate and ascorbic acid 2-phosphate; Neurogenic: DMEM, FBS, 2-mercaptoethanol, sodium pyruvate, all-trans retinoic acid and FGF-4; Pancreatic: DMEM, FBS, 2-mercaptoethanol, sodium pyruvate and nicotinamide on collagen I coated plate; Hepatic: DMEM, FBS, 2-mercaptoethanol, sodium pyruvate, dexamethasone, insulin, 1 phenobarbital on collagen I-coated plate.[194]
Table 8. Isolation, characterization, and differentiation of human amnion mesenchymal cells
Isolation protocolCharacterizationDifferentiation protocolReferences
  1. α-MEM = α-modified Eagle's medium; FBS = Fetal bovine serum; DMEM-H = Dulbecco's modified Eagle's medium high; TGF = transforming growth factor; IBMX = isobutyl-methylxanthine; EDTA = ethylenediaminetetraacetic acid; DMEM = Dulbecco's modified Eagle's medium; SH3 = Src homology 3 domain; SH4 = Src homology 4 domain; SH2 = Src homology 2 domain; BSA = bovine serum albumin; FGF = fibroblast growth factor; MCDB-201 = molecular, cellular, and developmental biology medium; VEGF = vascular endothelial growth factor; ITSLA = insulin, transferrin, selenium and linoleic acid.

The cells were digested with collagenase 2 and added in Minimum Essential Medium alpha (a-MEM, FBS).Positive cellular markers: CD13, CD49e, CD90, CD105, CD73, CD29, CD44, and CD166. Negative cellular markers: CD34, CD45 or CD14.Chondrogenic: DMEM-H, Fetal bovine serum (FBS), insulin, TGF-β1 and fresh ascorbic acid; Osteogenic: DMEM-H, FBS, β-glycerophosphate, 1α,25-dihydroxyvitamin D3, ascorbic acid, and dexamethasone; Adipogenic; DMEM-H/FBS/dexamethasone/isobutyl-methylxanthine (IBMX)/indomethacin and insulin; Myogenic: DMEM-H/FBS/ hydrocortisone/ dexamethasone and horse serum; Neurogenic: DMEM-H/FBS and all trans retinoic acid;[286]
The cells were digested with trypsin-EDTA solution, collagenase IV solution and DNAseI in DMEM. The supernatant was neutralized with FBS, centrifuged and re-suspended in DMEM, FBS, penicillin and streptomycin.Positive cellular markers: CD166, SH3, SH4, (anti-CD73), CD44, CD29 and SH2 (anti-CD105). Negative cellular markers: CD34, CD45 and CD14Chondrogenic: DMEM containing L-ascorbic acid-2-phosphate, dexamethasone, insulin, sodium pyruvate, transferrin, selenous, acid, linolenic acid, BSA, proline and Transforming Growth Factor-3; Osteogenic: DMEM containing FBS, dexamethasone, glycerophosphate and ascorbic acid; Adipogenic: DMEM supplemented with FBS, dexamethasone, indomethacin, isobutyl-methyl xanthine (IBMX) and insulin; Myogenic: DMEM supplemented with FBS, dexamethasone, FGFβ, MCDB-201, ITSLA*BSA, Insulin like Growth Factor-1, ascorbic acid-2-phosphate and Vascular Endothelial Growth Factor; Angiogenic: DMEM with FBS and VEGF[287]
Table 9. Isolation, characterization, and differentiation of chorion mesenchymal cells
Isolation protocolCharacterizationDifferentiation protocolReferences
  1. MSCs = mesenchymal stem cells; α-MEM = α-modified Eagle's medium; FBS = fetal bovine serum; CD = cluster of differentiation; DMEM-H = Dulbecco's modified Eagle's medium high; TGF = transforming growth factor; IBMX = isobutyl-methylxanthine; EDTA = Ethylenediaminetetraacetic acid; HLA = human leukocyte antigen.

Chorion was degraded with collagenase 2 after mechanical and enzymatic (dispase II) removal of the layers. Chorion MSCs were added in α-MEM/20% FBS.Positive cellular markers: CD13, CD49e, CD90, CD105, CD73, CD29, CD44 and CD166. Negative cellular markers: CD34, CD45 or CD14.Chondrogenic: DMEM-H, insulin, FBS, fresh ascorbic acid and TGF-b1; Osteogenic: DMEM-H, FBS, ascorbic acid, 1α,25-dihydroxyvitamin D3, β-glycerophosphate and dexamethasone; Adipogenic; DMEMH/FBSdexamethasone/indomethacin/insulin and isobutyl-methylxanthine (IBMX); Myogenic: DMEM-H/FBS/hydrocortisone/dexamethasone and horse serum; neurogenic: DMEM-H/FBS and all trans retinoic acid[286]
The cells were washed in PBS, digested in trypsin-EDTA/ collagenase I and filtered. The cells were centrifuged, resuspended in α-MEM and cultured flasks.Positive cellular markers: CD105, CD90 and CD73. Negative cellular markers: CD45, CD11b, CD79a, CD34 and HLA DR.Chondrogenic: chondrogenesis differentiation basal medium containing ascorbic acid-2-phosphate and TGF-β1; osteogenic: osteogenesis differentiation medium containing ascorbic acid-2-phosphate, dexamethasone and recombinant human bone morphogenetic protein;[288]
Table 10. Isolation, characterization, and differentiation of human amniotic fluid stem cells (hAFSCs)
Isolation protocolCharacterizationDifferentiation protocolReferences
  1. EDTA = ethylenediaminetetraacetic acid; HLA = human leukocyte antigen; CD = cluster of differentiation; MHC = MAJOR histocompatibility complex; DMEM = Dulbecco's modified Eagle's medium; FBS = Fetal bovine serum; HGF = hepatocytes growth factor; FGF = fibroblastic growth factor; ITS = insulin-transferrin-selenium; DMSO = dimethyl sulfoxide; NGF = neurogenic growth factor; DMEM/F12 = Dulbecco's modified Eagle medium/nutrient mixture F-12; plus 1× N2 = N-2 Supplement;

Amniotic fluid samples were cultured in DMEM or in Amniomax in cover glass. After, they were detached using a trypsin/EDTA solution and cultured with Chang Media supplemented with Chang B and Chang C.Positive cellular markers: c-kit, OCT-4, SSEA-4, CD29, CD44 (hyaluronan receptor), CD73, CD90, CD105, MHC Class I (HLA-ABC) and some were weakly positive for MHC Class II (HLA-DR). Negative cellular markers: CD45, CD34 and CD133.Adipogenic: DMEM low-glucose medium, FBS, dexamethasone, 3-isobutyl-1-methylxanthine, insulin and indomethacin. Myogenic: plastic plates precoated with matrigel, DMEM, horse serum, chick embryo extract and penicillin/streptomycin. After, 5-aza-2'-deoxycytidine is added to the culture medium for 24 h. Osteogenic: DMEM low-glucose medium, FBS, penicillin/streptomycin, dexamethasone, β-glycerophosphate and ascorbic acid-2-phosphate. Endothelial: endothelial basal media-2 in gelatin-coated dishes and FGFβ was added to the culture every 2 days. Hepatic: matrigel-coated dish, DMEM, FBS, monothioglycerol, HGF, oncostatin M, dexamethasone, FGF4 and 1× ITS. Neurogenic: Step 1: differentiation process for neural-like cells: DMEM low-glucose, penicillin/streptomycin, DMSO, butylated hydroxynisole and NGF. After 2 days, the medium was replaced with Chang supplemented with NGF only. Step 2: differentiation process for specific dopaminergic cells: fibronectin-coated plate, DMEM/F12, plus 1× N2 and bFGF.[289]
Table 11. Clinical applications in liver diseases
Cell TypeAnimal modelVia application cellResultsReferences
  1. hAECs = human amnion epithelial cells; CCl4 = carbon tetrachloride; ALT = alanine transaminase; TNF = tumor necrosis factor; IL = interleukin; TGF = transforming growth factor; hAMCs = human amniotic membrane-derived mesenchymal stem cells; CP-MSCs = chorionic plate-derived mesenchymal stem cells; TP = transplanted; Non-TP = non-transplanted; Col I = type I collagen; α-SMA = α-smooth muscle actin; ICG = indocyanine green; GOT = glutamate–oxaloacetate transaminase; GPT = glutamate–pyruvate transaminase; TBIL = total bilirubin; AF-MSCs = Human amniotic fluid mesenchymal stem cells; NOD-SCID mice = non-obese diabetes-severe combined immunodeficiency mice; HPL = hepatic progenitor-like; IFN = interferon; AFSCs = amniotic fluid stem cells.

Human amnion epithelial cells (hAECs)Six-week-old male C57/BL6 miceCell were injected intravenously into immune competent C57/BL6 mice 2 weeks after exposure to CCl4Reduces Serum AL; Reduction Hepatocyte Apoptosis; Decrease Hepatic Inflammation; Reduction in TNF-α and IL-6; Reduce Hepatic Fibrosis Area and TGF-β Content;[221]
Humanamniotic membrane-derived mesenchymal stem cells (hAMCs)Four- to 6-week old C57Bl/6J miceCells were infused into the spleen at 4 weeks after mice were challenged with CCl4Ameliorated the CCl4-induced deterioration of liver function; Alleviated CCl4-induced liver fibrosis; Reduced CCl4-induced hepatic stellate cell activation; Suppressed hepatocyte apoptosis and promoted hepatocyte regeneration; Suppressed hepatocyte senescence; hAMCs engraft in CCl4 injured liver[290]
Chorion mesenchymal cells (CP-MSCs)Six-week-old male Sprague–Dawley ratsCP-MSCs (2[lowem]106) were directly transplanted into the right liver lobe of each CCl4-injured rat by injection at a depth of 5 mmCP-MSCs were engrafted successfully and that some CP-MSCs differentiated into hepatocytes; Markers of liver damage (Col I, a-SMA and albumin): Col I mRNA expression was significantly lower in TP rats than in Non-TP, mRNA expression level of a-SMA was significantly lower in TP rats and albumin mRNA expression was significantly higher in TP rats than in Non-TP animals; Greater lipid accumulation and stronger macrophage infiltration in non-TP rats than in TP rats; Collagen deposition was dramatically lower in TP rats than in Non-TP animals; TP rats also displayed an overall reduction and amelioration of bridging fibrosis connecting neighboring portal and central veins; Liver damage scores assigned according to four-tiered cirrhosis criteria were lower in TP rats; TP rats exhibited significantly higher ICG release the non-TP rats; the levels of GOT, GPT, and TBIL were significantly lower in TP rats than in non-TP rats;[291]
Human amniotic fluid mesenchymal stem cells (AF-MSCs)NOD-SCID miceCells were intravenously transplanted into CCl4-treated mice 24 h laterAF-MSCs have the ability to engraft into CCl4-treated livers and to improve liver function; HPL cell transplantation was even more effective than treatment with AF-MSCs and successfully downregulated the systemic inflammation, as determined by measuring serum levels of IL-10, IL-2, IFNγ and TNF-α; A number of cytokines were secreted from HPL cells only, including IL-12p70 and IL-10 which induce local and systemic downregulation of proinflammatory mediators[292]
Amniotic fluid stem cells (AFSCs)Eight-week-old male nude miceCells were injected into the tail veins of the miceAFSC transplantation exhibited earlier restoration of a normal histology; Pathological changes caused by CCl4 (necrosis, inflammatory cell infiltration, and degeneration) disappeared more rapidly in the AFSCs group; The livers of the CCl4+AFSCs group had a normal histological appearance 10 d after the injury; Presume that transplanted cells secrete growth factors or similar agents that regulate the metabolism and proliferation of host hepatocytes[293]
Table 12. Clinical applications in heart disease
Cell typeAnimal modelVia application cellResultsReferences
  1. hAECs = human amnion epithelial cells; hAMCs = human amniotic membrane-derived mesenchymal stem cells; hAMCs = human amniotic fluid mesenchymal stem cells; SD = Sprague-Dawley; AFS = amniotic fluid stem cells; rNU rat = Rowett Nude rat.

Human amnion epithelial cells (hAECs)Male athymic nude rats 8 weeks of ageCells were injected into center of the infarcted myocardiumAttenuation of the left ventricular dilation; Improvement cardiac function; Gain the left ventricular ejection fraction; Reduction of the infarct area;[294]
Human amniotic membrane-derived mesenchymal stem cells (hAMCs)Wistar rats (8 weeks of age) and F344 nude rats (6 weeks of age)Cells were injected into the myocardiumTransdifferentiation of hAMCs into cardiomyocytes; Significant increase in the left ventricular fractional shortening; The percentage of fibrosis area was significantly decreased by hAMC transplantation; Evidence of Tolerance;[295]
Human amniotic fluid mesenchymal stem cells (hAMCs)Twelve-week-old female Sprague-Dawley (SD) ratsCells suspension were injected into the infarcted area at two or three sitesCardiac differentiation in the in vivo experiments was verified; Observation of a newly formed cell mass from hAMC in the scar suggested that hAMC will be a suitable cell source for the treatment of myocardiac infarction; hAMC survived in the scar tissue for at least 2 months and underwent cardiac differentiation; Survival of hAMC in xenotransplantation suggests the low immunogenicity of the cells[296]
Amniotic fluid stem cells (AFS)Female Sprague–Dawley (SD) and rNU ratsCells were injected in the periphery of the damaged area in three distinct injection sitesSmall masses of cells of different size were found in subendocardial or intramyocardial regions of left ventricle close to the injection sites; Only traces of the original AFS cells inocula could be found in the cardiac wall; Displayed a marked infiltration of inflammatory cells especially evident in the AFS cell-graft área; In the absence of AFS cell transplantation, rats did not evidence a lymphocyte infiltration.[297]
Table 13. Clinical applications in neurological diseases
Cell typeAnimal modelVia application cellResultsReferences
  1. hAEC = human amnion epithelial cells; CD = cluster of differentiation; EAE = experimental autoimmune encephalomyelitis; MOG = myelin oligodendrocyte glycoprotein; Th1 = IFN = interferon; GM-CSF = Granulocyte Macrophage Colony- Stimulating Factor; TNF = tumor necrosis factor; TGF = transforming growth factor; PGE2 = prostaglandin E2; hAMC = human amniotic membrane-derived mesenchymal stem cells; SD = Sprague-Dawley; iNOS = inducible nitric oxide synthase; GFAP = glial fibrillary acidic protein; Dcx = doublecortin.

Human amnion epithelial cells (hAEC)Female C57BL/6 mice 8–12 weeks oldCells were administered intravenouslyhAEC infusion ameliorated autoimmune encephalomyelitis in mice; Examination of spinal cord showed no or minimal inflammatory cell infiltration and myelin loss; Reduction in the numbers of CD3 T cells and F4/80 monocytes/macrophages in the spinal cords from hAEC treated mice; Splenocytes from hAEC-treated mice proliferated significantly less than EAE control mice after MOG peptide stimulation; Produce less Th1 cytokine IFN-gamma and less inflammatory cytokines GM-CSF and TNF-α; hAEC utilize TGF-β and PGE2 for immunosuppression[298]
Human amniotic membrane-derived mesenchymal stem cells (hAMC)Female Sprague–Dawley (SD) ratsCells were injected into the brain os ratsSurvival of transplanted hAMCs in the ischemic rat brain; Migration and differentiation of transplanted hAMCs in the ischemic rat brain; hAMCs decrease the infarct volume in the ischemic rat brain; Behavioral recovery; Activation inhibition of caspase-3 and iNOS in the ischemic rat brain[299]
Human amniotic fluid mesenchymal stem cellsAdult male Sprague Dawley ratsCells were transplanted in the striatumSurvival and majority of the transplanted cells were found migrating towards the ischemic lesion border; Human cells mostly co-labeled with the astrocyte marker. GFAP, and these cells could be detected in rat brains up to 90 days from the transplantation; a few cells were stained by the immature neuron marker Dcx, mostly in the adjacencies of the injection site, and only in brains at 10 days from transplantation; No human cells were found positive to the neuronal marker b-tubulin III at any time[300]
Amniotic fluid stem cellsSwiss albino mice of either sexStem cells were injected intracerebroventricularly (i.c.v.) 3 days after the surgical procedures were performed on mice.Extent comparable to the neuroprotective effect of embryonic neuronal stem cells; Increase in neurological severity score in terms of the results of the motor tests, sensory tests, beam balance tests as well as the reflex tests individually and also in composite manner as compared to the control group significantly (P < 0.05), in a dose dependent manner; Reversed the focal cerebral ischemia-reperfusion;[301]
Figure 1.

Stem cells were obtained from diverse tissues and isolated with different protocols. Adipose-derived mesenchymal stem cells (ASC), umbilical cord blood (Umbilical Cord Blood Mesenchymal Stem Cell - UCB-MSC) and bone marrow (Bone Marrow-derived Stem Cell-BMSCs) can differentiate into osteoblasts, chondrocytes, cardiomyocytes, neuron, adipocytes, myocytes, hepatocytes, pancreatic, epithelial and endothelial cells (also related to angiogenesis). Differentiation process is induced by growth factors, hormones and organic molecules (see Tables 2-5 for detailed information). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

Clinical Trials

Several reports in animal models have shown the therapeutic potential of SC. For instance, rats have had their intervertebral disc regenerated with a MSC implant [84]. Additionally, therapies in canines have also been successful [85]. Myocardial infarction in rats has shown good results after human BM-MSC transplants [86]. Encephalomyelitis has been improved in mice, decreasing the inflammation and stimulating oligodendrogenesis [87]. Moreover, MSC infusion promoted benefits in stroke therapy in rats [88].

In vivo identification of transplanted MSCs in animal models is done, normally, by histopathological labeling, as well as by fluorescent in situ hybridization (FISH), which are extremely evasive methods [86]. Some in vivo monitoring methodologies for the dynamics, distribution and localization of transplanted MSCs used are, fluorescent probes or reporter genes such as green protein fluorescent (GFP), 5-bromodeoxyuridine (BrdU) labeling, or 111In-oxine [89-94]. There are even more sophisticated approaches that enable the in vivo visualization and monitoring of MSC migration. Magnetic resonance imaging (MRI) uses superparamagnetic iron oxide nanocomposites that are internalized by the MSCs [95-97]. More recently, gold nanotracers have been used to label MSCs that can be monitored by ultrasound, and fotoacoustic methods [98].

Clinical procedures in humans have been carried out for decades with autologous peripheral blood transplants. Kessinger et al. infused patients that suffered from hematologic malignancies with peripheral blood [99]. Thomas et al. treated 100 Leukemia patients with bone marrow transplants until the 1970s [34]. More recent studies have shown success cases of cellular therapy with MSCs. Patients with full-thickness articular cartilage defects in the patellae that were transplanted directly on the articular cartilage with collagen gel containing BM-MSCs, had the defect repaired [100]. In another case, a patient with spinal cord-injury was treated with UC-MSC [101]. However, patients with hepatic cirrhosis that received HSC infusion in the peripheral vein did not show satisfactory results [102]. That same group performed BM-MSC transplant via the hepatic artery and the results were better, bringing hope for liver regeneration therapy [103].

Adipose and Menstrual-Derived Mesenchymal Stem Cells

Adipose Tissue-Derived Mesenchymal Stem Cells

The adipose tissue is an abundant and accessible source of stem cells, which possesses the ability to differentiate in several cell types. The International Fat Applied Technology Society adopted the term “adipose tissue-derived stem cells” (ASCs) to identify the cell population isolated from the adipose tissue. This population is characterized as plastic-adherent and multipotent cells [104]. Of note, different nomenclatures can be found in the literature to designate this specific cell population isolated from the adipose tissue, e. g. “adipose-derived adult stem (ADAS) cells,” “adipose-derived adult stromal cells,” “adipose-derived stromal cells (ADSCs),” “adipose stromal cells (ASCs),” “adipose mesenchymal stem cells (AdMSCs),” “preadipocytes,” “processed lipoaspirate (PLA) cells,” and “adipose-derived stromal/stem cells (ASCs)” [104-107]. ASCs are mesenchymal stem cells, thus are progenitors of cell types derived from the mesoderm and are also stromal cells. In later passages, ASC cultures are homogeneous and exhibit fibroblastoid morphology [108].

The adipose tissue is composed mainly of fat cells organized in lobules, which contain mature adipocytes in more than 90% of the tissue volume, and a fraction of the vascular stroma (SVF), where pre-adipocytes, fibroblasts, smooth muscle cells, endothelial cells, resident monocytes/macrophages, lymphocytes, and ASCs are found [109, 110]. Indeed, different populations of multipotent stem/progenitor cells are all CD45-negative. A progeny relationship between MSC and pericytes exists as basis for the different stem cell phenotypes in adipose tissue [64]. The inferior abdomen adipose tissue is the region that contains the highest percentage of ASCs compared with other sites. Additionally, the fibrous conjunctive tissue, which contains whiter adipose tissue, has more elevated content of ASCs. Rodbell, in 1964, was the first researcher to describe the technique to isolate adipose tissue cells [111, 112]. In this report, fat blocks were removed from rats and were successively washed to remove hematopoietic cells in the tissue. Then, the tissue was incubated and digested with collagenase to release the cell population. Among them, mature adipocytes and ASCs were separated by centrifugation and found in the superior and inferior fraction, respectively. The final selection is achieved by the adherence of the stem cells to plastic, thus the remaining cells, such as peripheral blood cells, fibroblasts, periocytes, and endothelial cells are discarded while maintaining the culture conditions [111, 112] (Table 2, Fig. 1). Therefore, ASCs can be isolated by their capacity to adhere to plastic of tissue culture dish. The procedure with mice is similar [113]. After isolation, ASCs are typically expanded in minimum medium containing 10% fetal bovine serum in a monolayer culture in plastic. Resembling protocol was used in the isolation of ASCs human stem cells [114]. Human fat blocks were also used, but advances in the surgical procedures have simplified this process. The aspirated material removed during lipoaspiration is a great source of stem cells. Because of saline and anesthetic infusion during the surgical procedure, the lipoaspirated material is found in fluid form, which facilitates the cell separation process and the majority of the cells can be recovered [111, 112] (Table 2, Fig. 1). Such a methodology allows the recovery of 0.1 to 1 × 109 nucleated cells from 200 mL of lipoaspirated material, out of which 10% of these cells are ASCs. In vitro, ASCs exhibit a duplication time of 2 to 4 days, depending on the culture medium and the passage number [115]. Regarding senescence, there are controversies about the conservation of telomerase activity during successive passages. According to several studies, the activity of this enzyme is reduced with successive passages and the characteristics of the cells are no longer identical, even revealing mutations in human ASCs [111, 116].

Menstrual Blood-Derived Stem Cells

The human endometrium is a tissue which undergoes dynamic remodeling, where over 400 cycles of regeneration, differentiation, and bleeding occur during the reproductive period of the woman [117]. The menstrual blood contains fragments of the endometrium that is shed during menstruation. The endometrium is composed of two layers, the functional layer, which is always undergoing restructuration, and the basal layer, composed mainly of loose conjunctive tissue [118].

MSCs can be obtained from the uterus through several processes, including hysterectomy, diagnostic curettage and menstrual blood [119]. In 2007, Meng et al. described a new population of stem cells extracted from menstrual blood [120]. The isolation of these cells was carried out through the collection of menstrual blood in appropriate vessels containing antibiotics and heparin. The cells were then processed in an antibiotic/antimycotic cocktail (vancomycin, sodium cefotaxime, amikacin, gentamicin, and amphotericin B), washed with phosphate buffered saline (PBS) and isolated by centrifugation in a discontinuous density gradient and through adhesion to plastic [120]. After isolation, this cell lineage can be maintained in minimum supplemented medium. The morphology of cells isolated from the menstrual blood is typical of mesenchymal stem cells [118], they possess the adult stem cell-like characteristics of self-renewal, high proliferative potential in vitro, and more important the ability to differentiate towards diverse cell lineages in the induction media. These multipotent cells have the ability to differentiate in several functional cells including cardiomyocytes, respiratory epithelium, neuronal cells, myocytes, endothelial cells, pancreatic cells, neurogenic cells, adipose cells and osteocytes (see Table 5, Fig. 1). Cultured adherent cells obtained from menstrual blood expand rapidly; their duplication time is 18 to 36 h [121] and those cells can be maintained in vitro for more than 68 duplications without compromising surface marker expression or presenting karyotypic abnormalities [120]. Recently, Khanjani et al. explored the differentiation ability of MBSCs into hepatoytes, and demonstrated that, although the degree of differentiation were extremely dependent on the concentrations of specific factors (hepatocyte growth factor and oncostatin M), the developed cells expressed mature hepatocyte markers, such as albumin, tyrosine aminotransferase and cytokeratin-18 at the mRNA and protein levels [122]. They also showed functional properties of hepatocytes, including albumin secretion, glycogen storage, and cytochrome P450 7A1 expression [122] (Table 5, Fig. 1). Therefore, MBSCs-derived hepatocyte-like cells can potentially lead to a major step toward application of stem cell therapy for chronic liver diseases.

Overall, MBSCs are a unique cell population that can be safely isolated (non-invasive procedure) and provide an expandable source of stem cells from childbearing to aged women. Undoubtedly, the study of the characteristics of MBSCs will provide a new insight into the future treatment of various diseases.

Surface Markers of Adipose and Menstrual-Derived Mesenchymal Stem Cells

Adipose-Derived Mesenchymal Stem Cells

Several independent research groups have investigated the ASCs in the effort to identify specific cell surface markers (Table 2, Fig. 1). Therefore, the expression of CD105/SH2, CD73 and CD90 proteins, and the lack of expression of CD45, CD34, CD14 or CD11b, CD79a or CD19 and HLA-DR proteins have been provided evidence for ASCs [123]. CD73 is considered as a characteristic marker of mesenchymal cells, and has been reported in addition to CD29, CD44 and SH3, which are also specific molecular markers characteristic of MSC.

The expression of stromal-derived factor-1 (STRO-1), which is usually used as marker for bone marrow progenitor cells, has also been found in ASCs. In contrast, the expression of hematopoietic lineage markers such as CD31, CD34, and CD45 was not detected in these cells through flow cytometry or immunofluorescence assays [123]. Alterations in the expression profile can be attributed to timing and passage number. After two or more successive passages in culture, the characteristics of adhesion molecules, surface receptors and enzymes, extracellular matrix and cytoskeleton proteins, as well as proteins associated with the phenotype of stromal cells can be altered. Nevertheless, despite differences in the cell isolation and culture procedures, the immunophenotype of adipose-derived stromal cells is relatively consistent across diverse studies [124]. Successive ASCs passages may lead to higher expression levels of CD117 (c-kit), human leukocyte antigen-DR (HLA-DR), as well as stem cell makers, such as CD34. At the same time, decreased levels of expression of stromal cell markers such as CD13, CD29 (β1 integrin), CD44 (hyaluronate), CD63, CD73, CD90, CD105 (endoglin) and CD166 have also been demonstrated. Additionally, the slight presence of CD106 has been reported in cultured ASCs [125]. A further study has shown that, after the third passage, the expression of OCT4, c-kit and CD34 in cultured ASCs is abolished, while Sca-1 expression has a significant enhancement. These cells have been identified as positive for CD29 and CD44, and negative for CD45 and CD31 expression [113]. Finally, in addition to the surface markers mentioned above, smooth muscle β-actin, platelet-derived growth factor (PDGF) receptor-β and neuro-glial proteoglycan 2 have also been identified in human adipose-derived stromal cells [126].

Menstrual Blood-Derived Stem Cells

Stem cells isolated from menstrual blood express typical mesenchymal stem cell markers, such as CD29, CD44, CD73, CD90, CD105, CD1, [42], which are also expressed in other tissues such as bone marrow and adipose tissue [121]. However, these cells do not express stromal-derived factor-1 (STRO-1), CD31 (epithelial cell marker), CD34 (epithelial and hematopoietic stem cell marker), CD45 (leukocyte marker), or HLA-DR [121]. Further studies have shown the expression of other surface proteins in this cell lineage by flow cytometry such as CD9, CD41a, CD59, and the lack of expression of CD14, CD38 and CD133, in addition to the already described markers [120]. Importantly, some authors have also reported the expression of pluripotency markers in MBSCs, such as octamer-binding transcription factor 4 (OCT4), stage-specific embryonic antigen 4 (SSEA-4), Nanog, and c-kit (CD117), in addition to the surface proteins [120, 127] (Table 5).

Clinical Applications

Adipose-derived stem cells

In the field of regenerative medicine, basic research and preclinical studies have been conducted to overcome clinical shortcomings with the use of adipose-derived mesenchymal stem cells. Using these attractive cell populations, researches have explored the safety and efficacy of implanted ASCs in different animal models. Likewise, preclinical data and ongoing clinical trials ASCs have been initiated in a variety of medical fields (Table 6).

It is known that current therapeutic approaches for muscle loss cannot restore muscle function effectively. Therefore, ASCs can be induced to differentiate into skeletal muscle cells and smooth muscle cells in vitro, which may provide an accessible and expandable alternative cell source for the cellular therapy of muscular disorders. Indeed, Di Rocco et al. demonstrated that ASCs have also shown a capacity for myogenic differentiation in vivo [128]. Allogeneic ASCs injected intravenously or directly into the affected muscle could restore muscle function in a murine muscular dystrophy model without any signs of immune rejection. In another study, the combination of ASCs induced to myogenic differentiation and injectable polylactic-co-glycolic acid (PLGA) spheres attached to myogenically-induced ASCs were injected subcutaneously into the necks of nude mice. They observed newly formed muscular tissues under the skin of mice that received ASCs induced to myogenic differentiation attached to PLGA, but not in those who received PLGA spheres alone [129]. However, it is still unclear whether ASCs directly differentiate into myogenic lineage cells or whether they become incorporated into muscle fibers via cell fusion. According to Di Rocco et al., it is likely that ASCs contain different subsets of cells capable of either function [128]. Enhancement of myogenic and muscle repair capacities of human adipose-derived stem cells with enforced expression of MyoD, by means of viral transductions, was also achieved in mice [130].

ASCs can also form osteoid cells in vivo. Osteogenic-induced ASCs cultured within atelocollagen honeycomb-shaped scaffolds with a membrane seal (ACHMS), when implanted subcutaneously in nude mice, possess high ability to differentiate into osteoblasts [131]. In this way, ASCs combined with different kinds of biomaterials or biomimetic composites were successfully employed in in vivo studies to repair critical bone defects in rabbit and murine models [132-134]. Also, human ASCs genetically modified by adenoviral gene transfer to overexpress BMP-2 (osteoinductive factor) could induce bone formation in vivo and heal a critically sized femoral defect in nude rats [135]. Short-term in vivo studies using ASCs in a goat spinal inter-body fusion model has also been reported [136]. Recently, Uysal and Mizuno demonstrated that topical injections of ASCs in the site of injury accelerates tendon repair in rabbits, as exhibited by a significant increase in tensile strength, direct differentiation of ASCs toward tenocytes and endothelial cells, and increases in angiogenic growth factors [137].

ASCs are promising candidate for myocardial regeneration, especially in acute clinical settings. Preclinical studies in large animals indicate that ASCs may be a potential alternative in cardiac cell therapy, since they are able to differentiate into cells presenting cardiomyocyte or endothelial phenotypes, and also express angiogenic growth factors and anti-apoptotic factors [138]. Accordingly, several in vivo studies have shown that allogeneic and xenogeneic transplantations of isolated ASCs have the potential to improve cardiac function in experimentally induced myocardial injury [139-148] (Table 6).

Human ASCs can spontaneously differentiate into cardiomyocytes in vitro, where vascular endothelial growth factor (VEGF) plays a critical role to induce differentiation; these cells express cardiac-specific markers troponin-I, myosin light chain 2 and show spontaneous contractions [149-151]. However, the in vivo differentiation of human ASCs into cardiomyocytes is still uncertain. Cai et al. found that intramyocardially-injected human ASCs differentiated into smooth muscle cells but not into cardiomyocytes in rats [139]. Similar observations were published by Rigol et al. [152]. Controversially, Bai et al. observed that fresh and cultured human ASCs when injected into the peri-infarct region of myocardial infarction-induced SCID mice, resulted in significant myocardial function [153]. Vascular density was significantly increased, and fewer apoptotic cells were present in the region of cell-injected. Immunofluorescence assay also revealed that grafted human ADCs underwent cardiomyogenic differentiation pathway. While the majority of studies have delivered ASCs by direct injection into the myocardial tissue, investigators have continued to explore the use of epicardial delivery via scaffold-free cell sheets. The advantages of these cell sheets is the absence of foreign material, the preservation of cell cohesiveness, and the possibility of incorporating different cell populations [154, 155]. Several types of cell sheet-based patches have improved damaged heart function in rat, canine, and porcine models [156]. In studies using rhesus monkeys, sheets of autologous ASCs provided a matrix for the delivery of allogeneic rhesus embryonic stem cells that had been differentiated along the cardiomyogenic lineage. A total of 2 months after the myocardial infarction (MI), the presence of the ASCs had improved angiogenesis. The study supported the safety of the ASC and embryonic stem cell combination [154]. In addition to this, many clinical trials of ADCs cell injection therapy have been performed and the controversial aspects about their propriety will be settled in next years, as more data is available.

The potential of adipose-derived cells to treat hindlimb ischemia or stroke has been investigated in several models as well. Transplantation of human ASCs cultured as spheroids and preconditioned under hypoxic conditions was found to improve recovery from hindlimb ischemia in murine models [157]. Additional studies have determined that ASCs exposed to ischemia or hypoxia secrete cytokines that can improve cell proliferation and vasculogenesis directly, without the presence of the ASCs themselves [158]. Finally, recent studies have evaluated the efficacy of autologous ASCs in a rat model of cerebral ischemia [159]. When ASCs were induced to undergo neuronal differentiation upon transplantion, the rats displayed improved neurological recovery and reduced infarct size compared with the controls [159].

The employ of ASCs is expanding to both the ectodermal and endodermal lineages. A study testing the effects of ASCs, where cells were differentiated into a Schwann cell-like phenotype, on peripheral nerve healing has been reported by di Summa et al. [160]. However, more experimental data must be gathered using larger animal models before these methods can be safely tested in the clinic.

ASCs can be used not only in peripheral nerve injuries but also in central nervous system injuries [161-165]. Ryu et al. demonstrated functional recovery and neural differentiation after transplantation of allogeneic ASCs in a canine model of acute spinal cord injury [165]. Immunohistochemical assessment indicated that the implanted ASCs differentiated into astrocytes and oligodendrocytes, as well as neuronal cells. In another study, ASCs were used for spinal cord injury in rats after in vitro differentiation into Schwann cells [163]. They speculated that neurotrophic factors derived from the grafted cells might have contributed to the promotion of functional recovery. In summary, it has been well established that ASCs can survive in the nervous system after injection and promote nerve healing either by direct differentiation or through the secretion of a number of paracrine factors. Thus, ASCs show promise for treatment of central nervous system and peripheral nerve related injuries in the near future.

The regenerative medicine also holds promise for the development of stem-cell-based therapy for the liver. Indeed, the in vitro induction of ASC differentiation can lead to the achievement of modified cells similar to hepatocytes, which present several liver-specific markers and related functions, such as albumin production, low lipoprotein uptake and ammonia detoxification [166]. More importantly, ASCs derived hepatocyte-like cells, after transplantation, were able to incorporate into the liver parenchyma of recipient mice [167]. Liver injury repair may also be possible with transplantation of rat ASCs, decreasing key liver enzyme levels and increasing serum albumin [168]. A yet controversial topic is the existence of hepatic stem cells discussed in a recent review [64].

Even diabetes can be treated by ASC therapy; murine ASCs transfer reduced hyperglycemia in diabetic mice [169, 170]. Based on all in vitro and in vivo research results scientists (Table 6) have investigated and explored different approaches in humans to employ ASCs in various clinical fields (Table 6).

Furthermore, breast reconstruction and augmentation trials have been reported by Yoshimura et al. [171]. Autologous adipose-derived stem cells were used in combination with lipoinjection in over fifty patients. The results showed no evidence of fibrosis or adhesions and improved fat grafting by the stromal vascular fraction (SVF) cells with retention of volume for over 12 months. Furthermore, a clinical trial was conducted for facial lipoatrophy using the same technique [172]. The authors noted improved facial contour, although there was no statistically significant difference in clinical improvement score compared with the conventional lipoinjection. Recently, Karaaltin et al. introduced the application of a successful ASC therapy for a linear scleroderma “en coup de sabre” deformity [173]. Although the patient's 1-year result demonstrated an improvement in appearance that required a refinement session of autologous fat grafting, the regenerative cell-enriched autologous fat grafting technique has provided a substantial enhanced result in one session of treatment [173].

In addition, there are some clinical experiences with bone reconstruction using expanded ASCs. Recently, restoration of human large bony defect using ASCs was reported [174-176]. Widespread calvarial defect was successfully repaired after autologous transplantation of SVFs in combination with fibrin glue [174]. Implantation of autologous cultured ASCs with β-tricalcium phosphate granules (β-TCP) was also reported in four patients who had large calvarial defects of different etiologies. Three months after operations, computed tomography scans revealed satisfactory outcome in ossification [176]. Mesimaki et al. published a clinical case report of prefabricated bone tissue engineering using autologous cultured ASCs with β-TCP and BMP-2 that resulted in successful maxillary reconstruction after bone flap transplantation in a patient submitted to a hemimaxillectomy [175]. A recent report of two clinical cases also demonstrated that treatment using ASCs in association with calcium chloride-activated platelet rich plasma and hyaluronic acid leads to the regeneration of medullary bone-like tissue and long-term reduction of hip pain in patients with femoral head osteonecrosis [177].

Furthermore, ASCs hold great promise for the treatment of human cardiovascular diseases. Currently, ongoing clinical trials using ASCs for cardiovascular treatment has been reported. First clinical trial is being carried out in 36 patients with end-stage coronary artery disease not amenable for revascularization and with moderate to severe left ventricular (LV) dysfunction to receive freshly-isolated ASCs via transendocardial. Second clinical trial is a study aimed to investigate the effect of ASCs on 48 patients with acute myocardial ischemia and LV ejection fraction impairment after appropriate infarct-related artery repair with stent implantation. In this study, freshly isolated ASCs will be delivered through intracoronary infusion within 36h following the onset of heart attack. Results from 14 patients show that ASCs were able to improve cardiac function of ischemia patients. At 6 months, an improvement of LV ejection fraction and reduction in infarct size in the ASC-treated group [178]. Now, a phase II/III ADVANCE trial has been initiated to evaluate their efficacy (http://www.clinicaltrials.org/NCT01216995/). A number of issues such as appropriate type and number of cells, timing and route of cell delivery, and the detailed mechanism of action should be optimized for more consistent clinical results [149].

On a different aspect, recently, Agorogiannis et al. in a case report presented a patient with post-traumatic persistent sterile corneal epithelial defect treated with topical application of autologous adipose-derived mesenchymal stem cells [179]. Corneal epithelial healing progression was started 11 days after topical application of autologous ASCs. One month later, a complete corneal epithelial healing was observed [179].

ASCs were also used to heal chronic fistulas in Crohn's disease [180, 181]. This disease is an inflammatory bowel disorder characterized by bloody stools, diarrhea, weight loss, and autoimmune-related symptoms. In a phase I trial with patients with fistulas unresponsive to standard treatment, cultured ASCs were directly injected into the rectal mucosa, and 75% of cases healed completely. In a phase IIb trial, the proportion of patients who achieved fistula healing was significantly higher with ASCs than with fibrin glue alone [182].

Although a recent review published by Locke et al. emphasized that the literature revealed considerable uncertainty about the true clinical potential of adipose-derived stem cells [183], several clinical trials are been passed on to patients around the world [184]. A search performed on www.clinicaltrials.gov (U.S. governmental web site maintained by the National Library of Medicine at the National Institutes of Health, NIH), with the search term “adipose stem cell therapy,” performed in June of 2013, revealed 80 open studies based on adipose-derived stem cell therapy widely distributed in numerous health and medical fields, which demonstrates the rapid evolution and expansion of clinical use of adipose-derived stem cells (Supporting Information Table S1).

Menstrual Blood-Derived Stem Cells

As mentioned above, menstrual blood-derived stem cells can rapidly expand and differentiate under laboratory conditions. These multipotent cells have the ability to differentiate into several functional cells including cardiomyocytes, respiratory epithelium, neuronal cells, endothelial cells, pancreatic cells, myocytes, hepatocytes, adipose cells and osteocytes [120]. Consequently, several recent studies have explored MBSCs in vivo regenerative potential to treat a variety of diseases (Table 6).

Duchenne muscular dystrophy (DMD), the most common lethal genetic disorder in children, is an X-linked recessive muscle disease characterized by the absence of dystrophin at the sarcolemma of muscle fibers. Cui et al. investigated menstrual blood-derived cells to determine whether these primarily cultured nontransformed cells would be able to repair muscular degeneration in a murine mdx model of DMD [185]. Transplantation of menstrual blood-derived cells directly into dystrophic muscles of immunodeficient mdx mice restored sarcolemmal expression of dystrophin. They also demonstrated that menstrual blood–derived cells can transfer dystrophin into dystrophied myocytes through cell fusion and transdifferentiation.

Hida et al. demonstrated that menstrual blood stem cell therapy can help repair damaged tissue [186]. Notably, MBSCs appear to be a potential novel, easily accessible source of material for cardiac stem cell-based therapy. After inducing differentiation these derived cells present a higher cardiomyogenic potential than those available from bone marrow. Additionally, after transplantation, MBSC-derived cardiomyocytes significantly restore impaired cardiac function, decreasing the MI area in a nude rat model. Also, transplanted cardiomyocytes could be observed in vivo in the MI area [186] (Table 6). In a subsequent study from the same group, Ikegami et al. claimed that they have established a fetal bovine serum-free cardiomyogenic transdifferentiation assay system in vitro [187]. They confirmed that the induction efficiency was greatly improved and was surprisingly at a higher level compared with that in serum-containing medium to generate MBSC-derived cardiomyocytes [186].

In order to test the therapeutic potential of menstrual blood-derived stem cells, Borlongan et al. (2010) used the in vitro stroke model of oxygen glucose deprivation (OGD) and found that OGD-exposed primary rat neurons that were co-cultured with menstrual blood-derived stem cells or exposed to the media collected from cultured menstrual blood exhibited significantly reduced cell death [188]. Trophic factors, such as VEGF, BDNF, and NT-3, were upregulated in the media of OGD-exposed cultured menstrual blood-derived stem cells. In addition, transplantation of menstrual blood-derived stem cells, either intracerebrally or intravenously and without immunosuppression, in an experimentally induced ischemic stroke in adult rats also significantly reduced behavioral and histological impairments compared with vehicle-infused rats. Therefore, such neurostructural and behavioral benefits afforded by transplanted menstrual blood-derived cells support their use as a stem cell source for cell therapy in cerebrovascular accidents (Table 6).

Although, in vivo studies in humans are at a preliminary phase, menstrual blood-derived regenerative cells were tested in four patients diagnosed with multiple sclerosis through intravenous and intrathecal injection of allogeneic endometrial cells. In this report, the case with the longest follow up (more than 1 year) revealed no immunological reactions or treatment associated adverse effects suggesting the feasibility of clinical endometrial regenerative cell administration and support further studies with this novel stem cell type [189]. In this context, ongoing phase I/II clinical trial investigates the safety and feasibility of using Endometrial Regenerative Cells, derived from menstrual-blood in patients, with critical limb ischemia that are not eligible for surgical or catheter-based interventions. The hypothesis is that endometrial regenerative cells administration will be well tolerated and possibly induce a therapeutic benefit (ClinicalTrials.gov Identifier: NCT01558908). Much still needs to be done and investigated to fully identify the superiority of MBSCs for basic research and clinical applications (Table 6).

Extra-Embryonic Tissue-Derived Stem Cells

The main source of adult stem cells is the bone marrow mesenchymal cells. However, this has some limitations. The aspiration of bone marrow cells is an invasive procedure and it causes discomfort to the donor, in addition to the fact that the potential of these cells decreases with age [190] and the amount of cells in the adult marrow is reduced [5, 191, 192]. An alternative source that is free of these limitations and of ethical questions would be the placenta. Moreover, this tissue is easily accessible at low cost since it is discarded after birth, and originates similar mesenchymal stem cells to the ones in the bone marrow, with the ability to expand and to express functional properties [193].

The placenta is made-up of three layers: the decidua, of maternal origin, the chorion and the amnion, of fetal origin [194]. Due to its complexity, it was necessary to create minimum criteria to define the placental mesenchymal cells. Five of them were proposed: [1] Cells must be of fetal origin, with less than 1% maternal contamination; [2] cells must adhere to a plastic surface; [3] cells should develop fibroblast forming units; [4] cells must differentiate in one or more lineage types; [5] cells must possess a standard set of surface markers CD90, CD73, CD105, CD45, CD34, CD14 and HLA-DR [195, 196].

The following human cells can be isolated from the fetal placenta: amniotic mesenchymal cells, amniotic epithelial cells, chorionic mesenchymal cells and chorionic trophoblast cells [196]. There are several protocols for the isolation of these cells as depicted in Tables 7-9 (Fig. 2).

Figure 2.

Stem cells from diverse origins require different isolation protocols. Stem cells derived from (menstrual blood-derived stem cells, MenSCs), amniotic fluid (amniotic-derived mesenchymal stem cells, AmnSCs), and chorion (chorionic mesenchymal stem cells, ChSCs) can differentiate into osteoblasts, chondrocytes, cardiomyocytes, neuron, adipocytes, myocytes, hepatocytes, pancreatic cells, epithelial and endothelial cells (also related to angiogenesis). Stem cells differentiation induction depends on growth factors, hormones and organic molecules treatment (for more detailed information see Tables 7-10. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

Another attractive source of mesenchymal stem cells is the amniotic fluid [197]. The amniotic fluid is found in the amniotic cavity, possesses an aqueous constitution and light color, allows the exchange of chemical products with the mother, protects the fetus against severe lesions, and allows the free movement and growth of the fetus within the cavity [198].

Until approximately the 1990s, cells capable of proliferating and differentiating in the amniotic fluid had not been reported. Torricelli et al. [199] used amniotic fluid collected before 12 weeks during pregnancy and found progenitor hematopoietic cells. Although Streubel et al. [200] showed non-hematopoietic precursors from the fluid differentiated into myocytes. These results suggest an alternative source for therapeutic applications [201].

Amniotic fluid cells can represent a bigger hope in regenerative medicine due to their easy access when compared with other extra-embryonic tissues. Two populations have been isolated from amniotic fluid so far: amniotic fluid mesenchymal stem cells, and amniotic fluid stem cells [201]. In order to isolate such cells, protocols use basically centrifugation of the samples and resuspension of the pellets in fetal serum and antibiotic containing medium, with further analysis of adipogenic and osteogenic potential, as described by some groups [197, 201, 202].

The surface antigen profile of these cells has been determined by flow cytometry. The amniotic fluid stem cells express markers such as SSEA-4, CD73, CD90, CD105, CD29, CD44, MHC-I and are negative for CD14, CD34, CD45, CD133, CD31, MHC-II [201, 203, 204]. On the other hand, the mesenchymal amniotic fluid stem cells are positive for CD90, CD73, CD105, CD166, CD29, CD44, CD49, CD54 and MHC-I and negative for CD45, CD34, CD14, CD133 and CD31 [197, 201, 205, 206]. Table 10 shows isolation, culture and induction protocol for amniotic fluid stem cells (Fig. 2).

Therapeutic Applications of Extra-Embryonic Tissue-Derived Stem Cells

Based on the characteristics described above, adult stem cells would be an alternative for therapeutic applications. The real situation of regenerative therapy can be analyzed in a report by Helmy et al. [207], which demonstrates the transplant of mesenchymal stem cells in 101 patients. This therapy can be performed by a autologous or allogeneic transplant through systemic or local infusion [208] and can be considered successful, when the following criteria are fulfilled: the cell must survive in the host after transplant, integrate in the tissue that needs repair, fulfill adequate function, have sufficient proliferation potential to repair the tissue and differentiate in the desired cell type and must not suffer graft reaction [209]. Recent reviews describe pathways involving the stem cells, its proliferation and differentiation [210-214]. However, the role of biologically active molecules produced by mesenchymal stem cells acting on target cells has been recently mentioned, changing the paradigm of the stem cell need to differentiate in specific tissue cells [211, 215]. Researchers studying Parkinson's disease and human epithelial amniotic stem cells believe that the observed beneficial effects are probably more related to these molecules than to the differentiation in neurogenic cells [216].

In the report by Meirelles Lda et al. [217], there are examples of these active molecules and they classified their paracrine effects as chemoattractants, immunomodulating, anti-scar forming and trophic, the latter being subdivided by mechanisms that prevent cell death or that induce precursor cell proliferation and differentiation, as well as the angiogenic effect.

Since it was thought that cell differentiation would be necessary for their in vivo application, several authors tested placental stem cells in vitro under particular culture conditions and observed the expression of neuronal and glial markers [194, 218]. Studies in non-immunosuppressed rats represent a significant evident of functional recovery from Parkinson's disease, as observed in reports by Kakishita et al. [219] and Okawa et al. [220].

Human epithelial amniotic stem cells possess hepatic genes and functions, representing an effective source for inducing differentiation into hepatic cells [196]. Mutations in those genes can result in metabolic disorders. Therefore, human epithelial amniotic stem cells may be an effective therapy, since these cells express the genes that are absent in the diseased liver [196]. Such therapy can be verified in hepatic and biliary fibrosis reports [221, 222]. To better understand these aspects, Tables 11-13 contains a list of clinical studies on hepatic, cardiac, and neurological diseases involving placental and amniotic fluid mesenchymal stem cells.

Induced-Pluripotent Stem Cells

Somatic cells can be reprogrammed to the pluripotent stage through the ectopic expression of transcription factors with many possible therapeutic applications in human disease (reviewed in Ref. [223]). Most studies have used fibroblasts as a starting population for the modification, but it takes weeks to achieve the ideal cell expansion. Such as lined at in the previous part of our review, MSC are especially promising for therapeutic approaches due to their broad differentiation capacity and due to their multi- or even pluripotency, therefore, undifferentiated cells form the perfect tool to reset them into the embryonic cells (reviewed in Ref. [224]).

As an example, ASCs can generate induced pluripotent stem cells (iPSCs). ASCs are easy to isolated and are found in large quantities, and can be easily maintained in culture [225]. Additionally, ASCs can be maintained in feeder-free conditions, thus eliminating the potential variability caused by the use of feeder cells [225]. Adipose tissue-derived stem cells intrinsically express high levels of pluripotency factors such as basic FGF, TGFp, fibronectin, and vitronectin, which can act as feeders for both autologous and heterologous pluripotent cells [226]. Human, rat, or mice ASCs possess high potential for reprogramming into inducible stem cells [225-227]. Although adipose tissue is a great source of stem cells and can be expanded, the potential for its use in regenerative medicine has not been broadly explored [225].

Thus, in an attempt to develop an adequate source for studies with iPSC, menstrual blood-derived stromal cells were identified as great candidates. It was shown that MenSCs could be reprogrammed to the pluripotent state through doxycycline-inducible lentiviral transduction of OCT4, SOX2, and KLF4. The resulting MenSC-iPSCs showed the same characteristics of human embryonic stem cells; morphology, pluripotency markers, gene expression, and epigenetic gene states. These cells were not only able to differentiate into several cell types of the three germ layers in vitro as well as in vivo, but also can be used for the generation of iPSCs [228, 229].

Nanog as a Mediator of the Pluripotent State in Mesenchymal Stem Cells

The transcription factors Oct4, Sox2, and Nanog present a central role in stem cell transcriptional network and maintenance of pluripotency (reviewed in Refs. [230] and [231]). Two others distinct sets of growth factors represented by LIF and Bmp4 versus bFGF and ActivinA, which are upstream on pluripotency state signaling cascade of Oct4, Sox2, and Nanog, signal to define and maintain two discrete pluripotent states termed naïve and primed stem cells. In mouse stem cells, two functionally distinct pluripotent states: a “naive” LIF-dependent pluripotent stem cell (PSC) state that is compatible with the preimplantation from the inner cell mass (ICM) and a “primed” bFGF-dependent PSC state that is reminiscent of the postimplantation epiblast [232]. The naive pluripotent state represents a more primitive stem cell population than the primed PSCs, in that they are uniquely capable of integrating with the blastocyst ICM and contribute to chimera formation. Between the three main factors, only Nanog has been shown to be a downstream target of both sets LIF and Bmp4 versus bFGF and ActivinA pathways that turns Nanog an attractive candidate as a key regulator of the pluripotent stem cell state.

Two independent groups looking for genes that can bestow LIF-independent growth on mESCs identified Nanog as the main transcription factor involved [233, 234]. In Nanog knockout studies it was shown that Nanog is not required for maintaining pluripotency, indeed Nanog−/− ESCs were revealed to be more prone to differentiation [235]. Furthermore, in the absence of Nanog, the derivation of naive PSCs is impaired, which is in according with the demonstration that Nanog has an important role in establishing a pluripotent state [236]. Recently, it was shown that several signaling pathways emanating from the LIF and ActivinA/TGF-β receptors regulate Nanog expression. Niwa and collaborators demonstrated that in mESCs, LIF-induced activation of PI(3)K/AKT induces T-Box 3 (Tbx3) expression, which in turn activates Nanog transcription (Fig. 3) [237]. Furthermore, parallel activation of the Jak/Stat3 pathway induces Klf4 expression; furthermore, binding to the Nanog promotor simulates its own expression [237, 238]. It was discovered that the human Nanog promoter has binding sites for SMAD2/3, which suggests that in human ESCs Nanog expression is direct influenced by ActivinA/TGF-β signaling. Indeed, ActivinA stimulation of human ESCs leads to an enhanced activity of the Nanog promoter, whereas mutations of the SMAD2/3 binding sites abrogate this responsiveness (Fig. 3) [239].

Figure 3.

Growth factors activating membrane cell receptors regulate Nanog expression levels in naive pluripotent stem cells. LIF stimulation results in activation of the PI(3)K/Akt, Stat3, and MEK/ERK Factor Signals, signaling pathways. Stat3 and PI(3)K/Akt positively regulate self-renewal: PI(3)K/Akt acts via T-Box3 and Stat3 acts via stimulating Klf4 expression. In contrast, activation of MEK/ERK signaling inhibits Nanog expression, but this pathway is inhibited by BMP4 signals through upregulation of Id proteins. Activation of PKA signaling by G protein-coupled receptors can stabilize the naive pluripotent state by maintaining Nanog expression levels through an unknown signaling mechanism. In primed pluripotent stem cells, ActivinA/TGF-β growth factor signaling induces Nanog expression via the SMAD2/3 binding site found in the Nanog promotor. Additional cooperative activation via bFGF was shown in human ESCs, but may not play a role in Nanog expression in murine EpiSCs. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

Interestingly, it is also possible to distinguish two populations within a mESC colony related to Nanog expression levels as Nanoghigh and Nanoglow. Even when Nanog-GFP mESCs are sorted into Nanoghigh and Nanoglow subpopulations heterogeneity is reestablished [235, 240]. The percentage of Nanoghigh cells is greatly increased under suppression of ERKs (extracellular-signal-regulated kinases) activation, which is critical for establishing and sustaining ES cells. In addition, inhibition of GSK3 (glycogen synthase kinase 3) reinforces this effects, demonstrating that high Nanog expression levels are closely linked to the inhibitor-enforced naive ESC state [241]. In contrast, Nanoglow cells within the ESC culture are more prone to differentiation, suggesting that low Nanog levels lead to a more primed pluripotent state [235], which can be related to the MSC pluripotency state in comparison to ESCs, suggesting their different differentiation capacity. Indeed, overexpression of Nanog facilitates the LIF-mediated conversion of epiblast stem cells (EpiSCs) into mouse embryonic stem cells (mESCs) [236]. These combined data suggest that the different sets of exogenous growth factors that support naive and primed PSCs promote a particular pluripotent state by maintaining different levels of Nanog expression. The recent generation of Nanog-GFP knock-in human ESCs demonstrates that Nanog exhibits heterogeneous expression in human ESCs as well, but it remains to be analyzed whether similar functional differences exist between NANOGlow and NANOGhigh human ESCs [242]. Besides some authors consider MSC plasticity as a consequence of cell culture conditions rather than an intrinsic MSC in vivo differentiation potential [243] it is reasonable that Nanog heterogeneity expression and maybe other transcription factor on downstream Nanog's cascade could control MSC plasticity. It was demonstrated that germ layer marker expression pattern would support the concept that MSCs possess plasticity, as it was shown by three-germ line marker co-expression and MSC capacity to differentiate into 3 germ lines (see Table 4 for differentiation protocols of MSCs). Recent worldwide survey conducted by International Stem Cell Initiative assessed expression pattern of stem cell associated markers in hES cell cultures. All hES cell lines tested exhibited a common expression pattern for a specific set of marker antigens and genes: glycolipid antigens SSEA-3 and SSEA-4, the keratan sulfate antigens TRA-1–60, TRA-1–81, GCTM2, and GCT343, and the protein antigens CD9, CD90, tissue-nonspecific alkaline phosphatase, as well as Nanog, OCT4, TDGF1, DNMT3B, GABRB3, and GDF3 [244]. Studies in mouse and human cells indicate that OCT4 is a component of a network of transcription factors, including the homeobox protein Nanog and HMG box transcription factor SOX2, that cooperatively maintain pluripotency in ESCs [245]. Cell surface marker SSEA-4 is a good indicator for undifferentiated hES cells, but it has also been used to isolate embryonic stem cell phenotype and has been studied in detail with several markers that have been reported as embryonic stem cell (ESC) specific antigens, including OCT4, Nanog, SOX2, SSEA-4, and others [244]. Recent studies have shown that adult stem cells, including MSCs, may express ESC markers: SSEA-4 expression has been detected in bone marrow [246] and it was recently shown that differential expression of others surface markers in mouse BM-MSC subpopulations have distinct lineage commitment. Furthermore, expression of CD200 was characteristic for the clones with osteogenic potential, whereas SSEA4 marked adipogenic progenitors lacking osteogenic capacity, and CD140a was expressed in adipogenic cells independently of their efficiency for osteogenesis [247] in dental pulp stem cells [248] and human deciduous periodontal ligament stem cells [249]. Oct4 expression has been reported in bone marrow [250] and adipose tissue-derived stem cells [251], peripheral blood mononuclear cells [252, 253], dental pulp stem cells [254, 255], heart and liver cells [250], however, Nanog expression has been found in the bone marrow, heart and liver mesenchymal stem cells [256]. Moreover, transcription factor SOX2 is expressed in the bone marrow, neural tissues and sensory epithelia from the early stages of development [257-260].

Conclusions

With the potential to treat a wide range of disease, from organ damage to congenital defects, HSCs form the underlying basis of regenerative medicine. Studies estimate that 250 billion dollars a year will be saved alone in the United States by employing stem cell-based therapeutics. The impact will be noticed mainly in the cost of treatment for chronic and neurodegenerative diseases such as Parkinson's disease, spinal cord lesions, cardiovascular diseases, neurovascular accidents and diabetes.

In particular, adult SCs and iPSCs derived from patient tissues may not only be a source of cells for autologous regenerative therapies but also in studying mechanisms of disease, and in drug screening and toxicology tests that are crucial in new drug development. Moreover, stem cell industry is witnessing continuous advancements globally and hence, many products are underway. The widespread availability of stem cell therapies will not only help make the treatment affordable in the coming years but also pave the way for personalized medicine.

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