Stem cells, including mesenchymal stem cells (MSCs), have promising applications for tissue engineering and regenerative medicine (1–5). New biocompatible scaffolds have been developed under multidisciplinary initiatives, permitting cells to grow and differentiate in locus, and exposing them to cytokines and growth factors in preparation for in vitro and in vivo applications (6, 7). Stem cells for clinical applications must be purified from original tissues by minimally invasive procedures, and then safely transplanted into autologous or allogeneic hosts.
The major MSC containing tissues, such as bone marrow and adipose tissue, are promising sources of multipotent cells (5) for replacement of lost cells and reestablishment of cellular function. Such strategies have been used for treatment of hematological diseases over the past decade (4). A number of therapeutic possibilities for MSCs have been proposed for degenerative diseases of bone, muscle, and the nervous system, and specially for cardiovascular and heart diseases (8–12), where they have been shown to be effective in re-establishing blood flow in ischemic tissues (11, 13). This particular type of stem cell (also termed mesenchymal stromal cell) is believed to have a perivascular origin (14) and adheres to plastic surfaces in vitro, proliferates on culture plates, and differentiates into at least three different mesodermal lineages upon specific stimuli (bone, cartilage, and adipose tissues). Moreover, several reports demonstrate the differentiation potential of these cells into several phenotypes, including endothelial cells (15, 16), cardiomyocytes (17), and neuron-like cells (18).
Cytometry Applications in Identification and Characterization of MSCs
Multipotent mesenchymal stromal cells of nonhematopoietic origin were first characterized in bone marrow (18), but they also can be isolated from many other tissues (19) with minor tissue-specific differentiation capacity differences, including umbilical cord blood, adipose tissue, muscle and placenta. Cell cultures can be replicated with cells remaining in their undifferentiated stage. According to different authors, these cells stain positive for a long list of cell surface markers including CD9, CD29, CD44, CD54, CD61, CD63, CD71, CD90, CD97, CD98, CD99, CD105, CD106, CD146, CD155, CD166, CD276, and CD304, while staining for CD14, CD34, CD45, and CD133 is negative (see Table 1). However, some authors reported conflicting results for positive or negative immunostaining, including Zimmerlin et al. defining CD34 as a MSC marker in adult adipose tissue (30). Positive staining for CD9, CD29, CD44, CD73, CD90, and CD166 and negative immunostaining for CD14, CD31, and CD45 was observed following induction of MSC to adipogenic and osteogenic differentiation. Expression levels of CD63, CD73, CD112, and CD166 increased during differentiation into both adipocytes and osteoblasts, while CD98, CD105, and CD155 expression was downregulated (24).
Table 1. Immunophenotypic characterization of MSCs of different origins
| ||Cell Types|
|hMSC||hMSC||hMSC + placenta||hMSC + placenta||hASC||hASC||hASC||Synovial tissue||Umbilical cord||Umbilical cord||Dental pulp|
|CD9||X|| || ||X||X|| || ||X||X|| || |
|CD10|| ||X|| || ||X|| || || || || || |
|CD13|| ||X|| ||X|| || || || || || || |
|CD18|| || ||X|| || || || || || || || |
|CD29||X|| ||X||X||X||X||X|| ||X||X|| |
|CD34|| || || || || ||X||X|| || || || |
|CD38|| || || || || || || || || ||X|| |
|CD44||X|| ||X||X||X|| ||X||X|| || || |
|CD49 a,b,c,e,f|| || ||X||X||X|| || || || || || |
|CD51|| || ||X|| || || || || || || || |
|CD54||X|| ||X|| ||X|| || ||X|| || || |
|CD55|| || || || ||X|| || || || || || |
|CD56|| || ||X|| || || || || || || || |
|CD58|| || || ||X|| || || || || || || |
|CD61||X|| || || || || || || || || || |
|CD63||X|| || || || || || || || || || |
|CD71||X|| || ||X||X|| || || || ||X|| |
|CD73|| || || ||X||X|| ||X|| ||X|| ||X|
|CD90||X||X|| ||X||X||X||X||X||X|| ||X|
|CD97||X|| || || || || || || || || || |
|CD98||X|| || || || || || || || || || |
|CD99||X|| || || || || || || || || || |
|CD104|| || ||X|| || || || || || || || |
|CD105||X||X|| || ||X|| || || || || ||X|
|CD106||X|| ||X|| || || || || || || || |
|CD146||X|| || || || || || || || || ||X|
|CD155||X|| || || || || || || || || || |
|CD166||X|| ||X|| ||X|| ||X||X|| || || |
|CD276||X|| || || || || || || || || || |
|CD304||X|| || || || || || || || || || |
|CD324|| || ||X|| || || || || || || || |
|LNGFR|| ||X|| || || || || || || || || |
|HLA-I|| || || || || || || || || ||X|| |
|HLA-DR|| ||X|| || || || || || || || || |
|HLA-ABC|| || || ||X|| || || || || || || |
|STRO-1|| ||X|| || || || || || || || || |
|BMPRIA|| ||X|| || || || || || || || || |
|Integrin α11|| || ||X|| || || || || || || || |
|Integrin β5|| || ||X|| || || || || || || || |
|Integrin β7|| || ||X|| || || || || || || || |
|Integrin β8|| || ||X|| || || || || || || || |
|ABCG2|| || || || || || ||X|| || || || |
|Survivin|| || || || || || || || ||X|| || |
|Bcl-2|| || || || || || || || ||X|| || |
|CD3|| || || ||X|| || || || || || || |
|CD14||X||X|| ||X||X|| || || || || || |
|CD16|| || || ||X|| || || || || || || |
|CD19|| || || ||X|| || || || || || || |
|CD27|| || || ||X|| || || || || || || |
|CD28|| || || ||X|| || || || || || || |
|CD31|| || ||X||X||X||X|| || || || ||X|
|CD33|| || || ||X|| || || || || || || |
|CD34||X||X|| ||X|| || || || ||X||X||X|
|CD36|| || || ||X|| || || || || || || |
|CD38|| || || || || || || || || || || |
|CD45||X|| || ||X||X||X|| ||X|| || ||X|
|CD50|| || ||X|| || || || || || || || |
|CD71|| || || || || || || || || || || |
|CD102|| || ||X|| || || || || || || || |
|CD105|| || || || || ||X|| || || || || |
|CD106|| || ||X|| || || || || || || || |
|CD117|| ||X|| || || || || || || || || |
|CD133||X||X|| || || ||X|| || || || || |
|CD144|| || || || || ||X|| || || || || |
|CD243|| || || ||X|| || || || || || || |
|HLA-DR|| || || ||X|| || || || ||X||X|| |
|FLK-1|| || || || || ||X|| || || || || |
|MOPC-21, 27–35|| || || || || || || ||X|| || || |
|FLA-1|| || ||X|| || || || || || || || |
MSCs from adipose tissue are particularly relevant for therapeutic use due to their relative abundance and simple isolation procedure when compared with bone marrow stem cells (31). These cells are morphologically similar to skin fibroblasts, but can be distinguished by a long list of molecular markers and by their enhanced differentiation potential (31). The identification of adipose MSC is currently based on the presence of surface markers (Sca-1, CD13, CD29, CD44, CD49e, CD90, CD73, CD105, among others, despite none of these being specific by itself) and on the absence of hematopoietic (CD45, CD133, CD117) and endothelial (CD31, CD10) antigens (32). Expression of markers such as CD34, however, may be regulated under in vitro culture conditions (25, 31). Surface marker expression patterns are routinely determined by flow cytometry (FCM) for cell-type classification, but it should be noted that a MSC cannot be detected based on a single marker protein expression, and consecutive single-parameter measurements for determination of an expression pattern of various stem cell markers may suffer from artifacts due to a heterogeneous cell population. Therefore, the Mesenchymal and Tissue Stem Cell Committee of the International Society for Cellular Therapy proposed three minimal criteria to define human MSCs: (i) MSCs must be plastic-adherent when maintained in standard culture conditions; (ii) MSC must express CD105, CD73, and CD90, and lack expression of antigens including CD45, CD34, CD14 or CD11b, CD79alpha or CD19, and HLA-DR surface molecules; (iii) MSC must differentiate into osteoblasts, adipocytes, and chondroblasts in vitro (33).
Even so, the application of the recommended markers for immunophenotyping is often not sufficient in heterogeneous cell mixtures such as the processed lipoaspirate (PLA). A general method for detecting adipose-derived stem cells is based on CD34+/CD31− immunolabeling. Blood vessel endothelial cells label CD34/CD31 positive, while smooth muscle cells are CD34−/CD31− negative (34), and CD34/CD45-positive cells in the adipose tissue are immune cells. It is clear that MSCs cannot be detected and isolated based solely on CD34 expression. Yoshimura et al. analyzed human PLA cells by multicolor FCM detecting expression patterns of CD31 and CD34, in addition to further stem cell markers CD45, CD90, CD105, and the endothelial cell adhesion molecule CD146, and were able to differentiate adipose MSC (CD31−/CD34+/CD45−/CD90+/C105− or +/CD146− cells) from endothelial progenitors (CD34+, CD146+) and pericytes (CD34−, CD146+) (24). Table 1 summarizes antigen expression patterns of stem cells of various origins.
Besides being used for stem cell identification and subsequent extraction from heterogeneous mixtures (Fig. 1), FCM as been used to detect phenotypic changes during stem cell culture and expansion in vitro, such as illustrated by the identification and isolation of stromal vascular progenitors in adult human adipose tissue (Fig. 2) (30). Cytometry techniques are being developed for visualizing stem cell engraftment in damaged tissue. Quantitative, noninvasive methods for tracking stem cells and their differentiation status will be essential for the understanding and optimization of their therapeutic effects of cell transplantation. Magnetic resonance imaging (MRI) is primarily used for the localization of stem cells, and can be coupled with positron emission tomography (PET) imaging for visualizing metabolic events (35). Alternatively, MSC can be labeled with the NIR815 membrane-intercalating dye followed by transplantation and near-infrared live imaging (36). More applications for stem cell imaging are still under investigation, and current imaging techniques mostly rely on the detection of expression levels of a reporter gene (luciferase) or green fluorescent protein (GFP) under the control of the regulation of a promoter of a differentiation-relevant gene. For instance, Lee et al. describe such reporter gene-expression imaging of specific differentiation markers for osteogenic differentiation of implanted MSC (37).
Figure 1. Flow cytometry for MSC characterization and purification for clinical applications. Tissue-specific multipotent stem cells, including MSCs, originated from pluripotent embryonic stem cells, remain in the adult body throughout the individual's lifetime, and are recruited for endogenous tissue repair. Among a considerable number of MSCs, adipose (ADSC) and bone marrow (BMSC)-derived cells are the most important ones for therapeutic applications and have been extensively characterized by immunophenotyping using FCM, i.e. for stromal cell markers CD29 and CD90 (see Supporting Information Table 1 for experimental details and MIFlowCyt-Compliance). Cell sorting can be used in addition to other separation techniques to obtain homogenous cell populations for differentiation assays and transplantation in clinical trials. MSCs are currently evaluated in clinical assays for various therapeutic applications, including bone repair and cell regeneration therapy in heart and neurodegenerative diseases.
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Figure 2. Classification of endothelial and perivascular populations for analytical flow cytometry. Top left box, upper row: identification of singlet cells with DNA content ≥2 N. Forward scatter (FS) pulse analysis was used to remove cell clumps remaining after disaggregation (A). Cells were gently permeabilized after fixation, permitting DAPI-dim hypodiploid events to be eliminated for the analytical population (B). Events with low light scatter were eliminated (C) on the basis of the location of small resting T lymphocytes (CD31+ CD45+ lymphocytes (Ly), color evented red). Top left box, lower row: elimination of autofluorescence. Cells with high autofluorescence form a diagonal streak in two-parameter histograms of the first three fluorescence channels. Events present in the compound gate of D and E and F were removed from analysis. In this sample, autofluorescent cells accounted for 8.1% of otherwise analyzable events. Right and bottom panel, top to bottom: classification of analyzable events into nonhematopoietic (NH) and hematopoietic (H), and nonhematopoietic endothelial (EM, EP) and nonendothelial (NE) populations. Within the endothelial mature subset (EM, pink) the majority of cells were CD90−, with some cells expressing CD146 and dim CD90. Endothelial progenitors (red) were exclusively CD90-bright with heterogeneous CD146 staining. A small proportion of CD90+ cells had saturating fluorescence and were eliminated from the analysis, not shown. Pericytes (Pe, orange) were defined among nonendothelial cells as CD146+, with a subset of bright CD90+ cells. A proportion of these CD90+ pericytes coexpressed CD34. SA-ASC (SA, green) was defined among nonendothelial cells as CD34+ CD146− cells. Like endothelial progenitors, the majority of SA-ASC coexpressed bright CD90. No CD117 expression could be detected on SA-ASC and other sorted nonhematopoietic populations as well (not shown). Light scatter profiles are presented for each candidate population. The region percentages represent the proportion of cells within each respective histogram (figure and legend correspond to Fig. 3 from Zimmerlin et al.) (30).
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“Musculoskeletal conditions comprise over 150 diseases and syndromes, which are usually progressive and associated with pain. They can broadly be categorized as joint diseases, physical disability, spinal disorders, and conditions resulting from trauma. Musculoskeletal conditions are leading causes of morbidity and disability, giving rise to enormous healthcare expenditures and loss of work” (World Health Organization)
The primary function of endogenous MSCs lies in the renewal and turnover of mesenchymal tissue including cartilage, bone, fat, and muscle. They are recruited when tissue injury occurs and provide an essential endogenous therapeutic tool. In addition to therapeutic strategies for enhancing recruitment of endogenous stem cells, cell engraftment is a treatment of choice for orthopedic disorders in which physiological endogenous tissue repair is impaired. Several studies have shown an efficient transplantation of MSCs for the treatment of diseases that affect musculoskeletal tissues, including osteogenesis imperfecta (OI), a genetic disorder for which there is currently no known cure. OI causes the osteoblasts to grow poorly, leading to bone deformities, bone fragility, and short stature. Some forms of OI may result in severe disability and even death. A preliminary study showed that intravenously infused bone marrow MSCs in children with OI migrate and differentiate into osteoblasts, contributing to improved bone structure (38). In a posterior study, severe OI was treated with competent donor bone marrow-derived MSC transplants resulting in increased body bone mineral content, growth acceleration, and a decrease in fractures rates (39). However, clinical follow-up showed that the growth rate slowed while bone mineral content continued to increase. Therefore, another clinical study was undertaken to evaluate the effect of an additional infusion of allogeneic MSCs. The results show that MSCs can engraft after transplantation and differentiate in osteoblasts, marrow stroma, and skin fibroblasts, and produce clinical benefits such as growth acceleration (40). Le Blanc et al. (41) performed intrauterine transplantation of allogeneic HLA-mismatched fetal MSCs in a 32-week fetus with severe OI. The obtained results indicate that the transplanted cells provided a source of osteoblastic progenitor cells, and the child had normal psychomotor development at 2 years of age. This report shows that allogeneic fetal MSCs can differentiate into bone in a human fetus even when the recipient is immune-competent and HLA-incompatible (41).
Whyte et al. in 2003 reported the first trial of bone marrow transplantation for hypophosphatasia, a rare metabolic bone disease caused by deficient activity of the isoenzyme tissue-nonspecific alkaline phosphatase (42). There is no established medical treatment for this disease. The patient was an 8-month-old girl who received T-cell-depleted bone marrow transplantation from her sister. Observed clinical improvement included enhanced bone mineralization, cure of rickets, and absence of new fractures. However, between 6 and 9 months after receiving the transplant the patient deteriorated clinically, and at 21 months age the patient received ex vivo expanded bone marrow MSCs. Significant, prolonged clinical and radiographic improvement followed soon after, suggesting that the observed results are due to the transient and long-term engraftment of sufficient numbers of donor marrow mesenchymal cells, forming functional osteoblasts and chondrocytes to improve her skeletal disease state (42). In another study, a 9-month-old girl suffering of infantile hypophosphatasia was treated with a heterogeneous cell population, including bone fragments, by three different routes (intraperitoneal, subcutaneous, and intravenous) (43).
Recently, Tadokoro et al. (44) related the outcome of a trial including allogeneic bone marrow cell transplantation and infusion of culture-expanded donor MSCs, a local injection of osteoblastic cells, and implantation of cultured osteoblastic cells on ceramics (osteogenic constructs). After treatment, there was gradual improvement of bone mineral density, respiratory condition, and body height and weight. Moreover, the retrieved construct showed de novo bone formation derived from both donor and patient cells (44).
Osteonecrosis occurs when bone loses its blood supply and dies. Pak (45) reported two cases of osteonecrosis, one in the femoral head and the other in the right hip. Patients' adipose tissue-derived stem cells were collected by a lipoaspiration procedure. Twelve weeks after transplantation, they observed a significant bone defect fill and probable matrix formation in both cases (45). Another application for MSCs is the treatment of fractures originating from nonunion, delayed union, and mal-union of bones. In these cases, the effects of locally injected autologous MSCs were evaluated. Quarto et al. (46) reported the case of three patients with large bone defects (4–7 cm) who had been treated with ex vivo expanded autologous MSCs. The cells were placed on macroporous hydroxyapatite scaffolds, whose sizes and shapes were consistent with the particular bone defect in each patient, and then implanted at the lesion site and stabilized with an external fixator. Abundant callus formation along the implants and good integration were observed 2 months after surgery, indicating transplantation success (46). Using the same approach, a study of four patients with up to 7 years follow-up showed that complete fusion between scaffold and bone recovery occurred after 5 to 7 months following surgery. No late fractures in the implant zone were observed suggesting, the long-term durability of bone regeneration achieved by a bone engineering approach (47). In a study of atrophic nonunion of the tibia, bone marrow cells were injected in the nonunion gap and around the bone ends. The authors concluded the safety and effectiveness of the trial; however, efficacy of stem cell engraftment was measured as the number of progenitor cells in the graft instead of considering the patient's health (48). Chondrocytes constitute the unique cellular component of cartilage. Articular cartilage is nonvascularized and noninnervated and has a limited capacity to repair itself. Surgical treatment is often necessary to encourage repair and, left untreated, local cartilage damage can result in osteoarthritis (OA). OA occurs with ageing and as a result of mechanical stress and inflammation, leading to primary focal cartilage degradation and its functional loss (49). MSCs seem promising for cartilage repair, since these stem cells naturally originate this tissue.
Many clinical studies are underway to evaluate therapeutic bone regeneration using MSCs from different sources and their application in different musculoskeletal diseases. Arthritis and cartilage defects and lesions are definitely among the most promising applications of MSC therapy, being already in use in sport medicine (see Table 2 for further examples).
Table 2. Clinical trials undertaken with MSCs in bone disease (data source: www://ClinicalTrials.org)
|Trial number||MSC source||Phases||Summarized disease|
|NCT00702741||Bone marrow||Phase I/II||Meniscectomy|
|NCT01420432||Umbilical||Phase I||Ankylosing spondylitis|
|NCT01159899||Bone marrow||Phase I/II||Arthorisis|
|NCT00891501||Bone marrow||Phase II/III||Arthritis|
|NCT00851162||Bone marrow||Phase II/III||Bone neoplasms|
|NCT00850187||Bone marrow||Phase I||Cartilage defect|
|NCT00885729||Bone marrow||Phase I||Cartilage defect|
|NCT01399749||Adipose||Phase I/II||Cartilage lesion|
|NCT01207193||Bone marrow||Phase I||Cyst|
|NCT01389661||Bone marrow||Phase I/II||Cyst|
|NCT01183728||Bone marrow||Phase I/II||Degenerative disease|
|NCT01183728||Bone marrow||Phase I/II||Degenerative disease|
|NCT00250302||Bone marrow||Phase I/II||Fractures|
|NCT01206179||Bone marrow||Phase I||Fractures|
|NCT01429012||Bone marrow||Phase I/II||Fractures|
|NCT01227694||Bone marrow||Phase I/II||Gonarthosis|
|NCT01210950||Bone marrow||Phase I||Leg length inequality|
|NCT00225095||Bone marrow||Phase I/II||Menistectomy|
|NCT01207661||Bone marrow||Phase I||Osteoarthritis|
|NCT01436058||Bone marrow||Phase I||Osteoarthritis|
|NCT01459640||Bone marrow||Phase II||Osteoarthritis|
|NCT00186914||Bone marrow||Phase I||Osteodysplasia|
|NCT00187018||Bone marrow||Phase I/II||Osteogenesis imperfecta|
|NCT00813267||Bone marrow||Phase I/II||Osteonecrosis of the femoral head|
|NCT00557635||Bone marrow||Phase II||Pseudo-arthosis|
|NCT01309061||Adipose||Phase II||Romberg's syndrome|
The recent discovery of a regenerative capacity in the adult central nervous system (CNS) by neural stem cells brings forth the possibility of repairing damage resulting from deteriorating neurodegenerative diseases such as ALS, Alzheimer's and Parkinson's diseases, and also brain and spinal cord injuries resulting from stroke or trauma. Recent efforts strive to recuperate brain tissue by inducing the patient's endogenous cells to differentiate and integrate into neuronal networks, but most approaches still rely on transplantation of stem cells. There are two basic strategies for cellular therapy of neurodegenerative diseases. One is to culture the stem cells and induce the desired differentiated neuronal cell type before implantation, the second is to implant them directly. In this case, endogenous factors secreted by the damaged tissue are expected to induce differentiation and finalize endogenous repair mechanisms.
Bone marrow stem cells have been shown to improve neurological performance of rats with brain ischemia (50). Recently published studies suggested that MSCs could generate specialized cell types derived from a different germ layer (51). Such transdifferentiation would be useful for cellular therapies in neurodegenerative diseases, because they sidestep some difficulties related with the use of embryonic stem cells, such as the potential for tumor formation, immunological rejection, and some ethical concerns (51–54), and the limitations in obtaining neural stem cells from brain during biopsies or surgery. Indeed, MSCs possess a notable phenotypic plasticity, and some studies suggest that MSCs may give rise to neural phenotypes (55–59). Other studies did not confirm neural differentiation of MSCs, but concluded that MSCs may fusion with existing neural cells at the site of transplantation (60). A widely accepted hypothesis holds that MSCs release neurotrophic factors which then recruit endogenous neural stem cells to the site of tissue repair (61). Although available evidence indicates that little CNS infiltration occurs after intravenous delivery of MSCs, making their effects likely peripheral (62, 63), some authors disagree (64).
The ability of MSCs to positively affect CNS repair has also been confirmed in experimental models of chemical demyelination (65, 66), cerebral stroke (50, 67), trauma (68), and Parkinson's disease (69, 70). Some data suggest that MSCs can be released into circulation from the bone marrow or other tissues and improve disease conditions by modulating a wide variety of immune responses (52, 71, 72). For instance, in an acute traumatic brain injury (TBI) model, MSCs modulated the inflammatory response by simply changing the expression of pro- and anti-inflammatory cytokines, together with serum level modulation of chemokines and other neuroprotective effects (73, 74). Walker et al. observed that MSCs implanted in TBI rats increased interleukin (IL)-6 concentrations (75), exerting both direct and indirect neurotrophic effects on neurons (76). In addition, MSCs are capable of both adaptative and innate immunity in vitro, corroborating their immunomodulatory role (77). Interestingly, MSCs also enhance remyelination in models of demyelination which do not involve inflammation (52, 78, 79). These data agree with results showing that MSCs could secret soluble factors for stimulating proliferation and differentiation of neural stem cells in vitro and in vivo (80–83). These features, together with the suggested ability of MSCs of transdifferentiation into neural cells and/or liberation of neurotrophic factors (76, 84) and migration to the central nervous system (85), have engendered clinical trials for evaluation of their therapeutic potential in neuronal repair.
Experimental autoimmune encephalomyelitis (EAE) is an animal model for human multiple sclerosis (MS), a common neurological disease and a major cause of disability, particularly in young adults. MS is an inflammatory disease, resulting in extensive multifocal demyelination and axonal loss throughout the CNS (86, 87), and is characterized by patches of damage occurring throughout the brain and spinal cord, with loss of oligodendrocytes and myelin sheaths. Although the cause of MS remains unknown, it is generally assumed that an autoimmune reaction against oligodendrocytes and myelin plays a major role, and early acute MS lesions almost invariably show prominent inflammation. Intravenous infusion of MSCs has been shown to improve the clinical course in EAE models (88, 89). Furthermore, MSCs injected on one side of the brain induced peripheral T-cell tolerance to myelin proteins, reducing migration of pathogenic T cells to the CNS, while on the other side of the brain MSCs homed themselves to the CNS where they preserved axons and reduced demyelination. It was also observed that MSC administration in EAE mice enhances remyelination, possibly through the release of neurotrophins such as brain-derived neurotrophic factor (BDNF) (81, 90, 91).
Another disease of interest for MSC-based therapies is Parkinson's disease, a progressive movement disorder. Early symptoms include uncontrollable hand tremor, followed by increasing rigidity and trouble initiating voluntary movement resulting from the death of nigro-striatal neurons, which release the neurotransmitter dopamine on striatum neurons and thus regulate the nerves that control body movement. Cell replacement therapies for Parkinson's disease have focused on delivering dopamine-producing cells to the striatum, and genetically engineered neural stem cells secreting neurotrophic factors showed promising results (92). However, there are limited sources of neural stem cells, and maintenance and differentiation protocols for these cells need yet to be defined (93, 94). Therefore, MSCs have been evaluated as source for the repair of the dopaminergic system. After chemical induction to neuronal differentiation, MSCs downregulate mesenchymal-lineage specific markers but fail to mature into functional neurons, as assessed by patch clamp studies. More experiments are necessary before MSCs can be considered a source for replacement of dopaminergic neurons (95).
Stroke is a common and disabling condition that represents an attractive target for regenerative therapy. Stem cells from diverse origins have been investigated in studies using animal models of stroke, and showed that neural or mesenchymal cells migrate to the site of ischemic injury after intravascular or intraparenchymal delivery, and that a proportion of cells survive and differentiate into cells with characteristics of neurons or glia. Some studies showed electrical activity of transplanted cells. Few clinical trials have been undertaken to date, with only two studies reporting intravascular delivery of bone marrow derived MSCs. On the first, MSCs were administered in a controlled trial to a small number of subjects (5 actively treated and 25 controls). The authors reported improvement in one functional score of activities of daily living (Barthel index scores measured at 3.6 and 12 months after cell therapy) but not in other clinical measures of outcome (modified-Rankin scale and National Institutes of Health Stroke Scale scores) (96). On the second trial, autologous cells were transplanted intraparenchymally in an open study, and clinical scores showed minimal and insignificant improvements (97). Intravenous infusion of MSCs in rats subjected to TBI failed to result in significant acute or prolonged cerebral engraftment of cells or to modify the recovery of motor or cognitive functions (98).
Clinical trials involving the use of human MSCs for the treatment of other neurological disorders, however, have shown promising results. Transplantation of autologous bone marrow MSCs by intrathecal injection into the spinal cord cerebrospinal fluid, allowing access to the CNS, was accomplished safely in patients with multiple sclerosis (MS) and ALS. Karussis et al. provided several lines of evidence of immunomodulatory effects of MSCs after intrathecal injection, but the MSCs that persisted after 3 to 6 months were less effective (99).
Spinal cord injury (SCI) results from damage to axons, loss of neurons and glia, and demyelination. Some studies showed that MSC administration, leading to grafting together with nestin-positive cells, can recover some of the clinical symptoms (68, 100, 101). Interestingly, a recent clinical trial revealed that patients with complete SCI who had received unmanipulated autologous bone marrow transplants 10 to 467 days following the injury showed improvement in motor and/or sensory functions (102).
Based on these studies, Mazzini et al. sought to verify the efficacy of MSC transplantation in patients with ALS. ALS is a pathology that causes a selective loss of motor neurons leading to a progressive decline in muscle functionality and poor prognosis. Current therapies only alleviate symptoms and there is no cure (103). The research group enrolled seven patients with ALS, showing severe lower limb and mild upper limb functional impairment. A bone marrow aspirate from each patient was used to prepare MSC cultures that were expanded for 3 to 4 weeks. Cells were then suspended in autologous cerebrospinal fluid and directly transplanted into the surgically exposed spinal cord at T7–T9 levels. No patient experienced severe adverse events following transplantations. MRI performed 3 and 6 months after transplantation did not show any structural changes of the spinal cord or abnormal cell proliferation when compared with the baseline. Three months after cell implantation, a mild trend toward a slowing down of muscular strength decline was observed in lower limb proximal muscle groups in four patients (104). These preliminary results are not sufficient for drawing any conclusions about the efficacy of MSC transplants for ALS treatment, but they pave the way for further studies and trials aiming to treat neurodegenerative diseases and other neurological disorders (see Table 3 for further examples).
Table 3. Clinical trials undertaken with MSCs in neural repair (data source: www://ClinicalTrials.org)
|Trial number||MSC source||Phases||Summarized disease|
|NCT01290367||Bone marrow||Phase II||Degenerative disc|
|NCT00549913||Bone marrow||Phase I/II||Degenerative disc|
|NCT00810212||Bone marrow||Phase I/II||Degenerative disc|
|NCT01297218||Umbilical||Phase I||Multiple sclerosis|
|NCT00813969||Bone marrow||Phase I||Multiple sclerosis|
|NCT01453764||Adipose||Phase I/II||Multiple sclerosis|
|NCT01325103||Bone marrow||Phase I||Nonunion fractures|
|NCT01453803||Adipose||Phase I/II||Parkinson's disease|
|NCT01446614||Bone marrow||Phase I/II||Parkinson's disease|
|NCT01453803||Adipose||Phase I/II||Parkinson's disease|
|NCT00976430||Bone marrow||Phase I/II||Parkinson's disease|
|NCT01228266||Bone marrow||Phase II||Sclerosis|
|NCT01051882||Bone marrow||Phase I/II||Sclerosis|
|NCT01377870||Bone marrow||Phase I/II||Sclerosis|
|NCT00781872||Bone marrow||Phase I/II||Sclerosis|
|NCT00395200||Bone marrow||Phase I/II||Sclerosis|
|NCT00962923||Bone marrow||Phase I/II||Sclerosis|
|NCT01364246||Bone marrow||Phase I/II||Sclerosis|
|NCT01142856||Bone marrow||Phase I||Sclerosis|
|NCT01446640||Bone marrow||Phase I/II||Spinal cord injury|
|NCT00816803||Bone marrow||Phase I/II||Spinal cord injury|
|NCT01274975||Adipose||Phase I||Spinal Cord injury|
Heart ischemic diseases are the leading cause of morbidity and mortality in the developed and in developing world (105, 106). Their pathological processes are due to obstructive lesions that prevent blood flow into the heart muscle, causing cardiomyocyte death and progressive deterioration of the organ. Mortality rates for 30 days, 1 and 5 years after the start of hospitalization are 10%, 22%, and 42%, respectively (107). Similarly to other degenerative diseases, treatment options are often insufficient to severe cases, due to limited regenerative capacity of heart (108, 109). Preclinical studies have tested the best conditions for cell delivery, such as cell injection routes (intravenous, coronary or intramyocardial injection), timing (acutely—up to 72 h—or chronically—several months after infarction) and doses (from millions to billions, depending on animal size) (110). Adequate dosage is particularly relevant, because frequently there are few cells remaining in the heart after injection, due to the washout effect (111) and cell death caused by hypoxia, low nutrient supply, and tissue strain (112). These difficulties were partially solved by using biopolymers, such as fibrin or collagen, as means to increase the retention of cells on tissues (113, 114). Furthermore, approaches based on transplantation of cells in three-dimensional sheets, harvested from temperature-responsive culture surfaces (115, 116), or MSC sheet fragments (117), made possible the creation of cell patches, further improving cell retention and viability.
The transplantation of both cultured and freshly isolated MSCs has provided strong evidence for attenuation of cardiac dysfunction in animals with myocardial injury (118). Moreover, the results of the completed clinical trials have demonstrated that stem cell therapy is safe and can improve recovery from myocardial ischemia (119). Today, at least 25 other trials of different phases are in progress, using these cells mainly in infarction scenarios (see Table 4, for further examples). However, differently from what had been believed in the beginning of the decade, the beneficial effects of simple transplantation of MSCs in cardiac disease could not be explained by their differentiation capability, particularly, into cardiomyocytes (120) and endothelial cells (121), nor by cell fusion (122, 123). At that time, a series of in vivo and in vitro differentiation strategies were giving support for MSCs ability to differentiate into cardiac cells (124–128) and possibly promote regeneration of heart, similarly to other stem cells types (129). Again, as already discussed for MSC roles in brain repair, their main mechanism of action is to liberate factors that modulate inflammation by suppressing lymphocytes activation (130, 131), and induce anti-apoptotic (132), cardioprotective (133) and, most importantly, angiogenic (134, 135) effects, orchestrating the repair by facilitating the formation of new vessels from pre-existing ones. In support of such hypothesis, injection of conditioned cell culture medium into the infarcted heart promoted a strong proangiogenic and antiapoptotic effect (136). Moreover, production of these factors is exacerbated in situations of hypoxia (137). The formation of new vessels promotes an increase of irrigation in the region of the ischemic heart, which in turn provides an abundance of nutrients and oxygen to reduce tissue damage and increase cardiac contractility (138). Furthermore, when MSCs were co-cultured with endothelial cells, they efficiently stabilized nascent blood vessels in vivo by functioning as perivascular precursor cells (139).
Table 4. Clinical trials undertaken with MSCs in cardiovascular and heart disease (data source: www://ClinicalTrials.org)
|Trial number||MSC source||Phases||Summarized disease|
|NCT01392625||Bone marrow||Phase I/II||Cardiomyopathy|
|NCT00629096||Bone marrow||Phase II||Cardiomyopathy|
|NCT00587990||Bone marrow||Phase I/II||Heart failure|
|NCT00644410||Bone marrow||Phase I/II||Heart failure|
|NCT00721045||Bone marrow||Phase II||Heart failure|
|NCT00768066||Bone marrow||Phase I/II||Heart failure|
|NCT00810238||Bone marrow||Phase II/III||Heart failure|
|NCT00927784||Bone marrow||Phase II||Heart failure|
|NCT01087996||Bone marrow||Phase I/II||Heart failure|
|NCT01442129||Bone marrow||Phase II||Heart failure|
|NCT00114452||Bone marrow||Phase I||Myocardial infarction|
|NCT00418418||Bone marrow||Phase II||Myocardial infarction|
|NCT00555828||Bone marrow||Phase I/II||Myocardial infarction|
|NCT00877903||Bone marrow||Phase II||Myocardial infarction|
|NCT00883727||Bone marrow||Phase I/II||Myocardial infarction|
|NCT01291329||Wharton's jelly||Phase II||Myocardial infarction|
|NCT01392105||Bone marrow||Phase II/III||Myocardial infarction|
|NCT01394432||Bone marrow||Phase III||Myocardial infarction|
|NCT00442806||Adipose||Phase I||Myocardial infarction|
|NCT00426868||Adipose||Phase I||Myocardial ischemia|
|NCT00260338||Bone marrow||Phase I/II||Myocardial ischemia|
|NCT00790764||Bone marrow||Phase II||Myocardial ischemia|
|NCT01076920||Bone marrow||Phase I/II||Myocardial ischemia|
|NCT01449032||Adipose||Phase II||Myocardial ischemia|
Therefore, even if MSCs are not capable of forming new heart cells, which would be necessary for heart regeneration in larger infarction volumes, they may provide an important clinical benefit to patients by preserving the heart from the devastating effects of a myocardial infarction.