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 . 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 . 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 . 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 .
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 . 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 . Short-term in vivo studies using ASCs in a goat spinal inter-body fusion model has also been reported . 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 .
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 . 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 . Similar observations were published by Rigol et al. . 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 . 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 . 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 . 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 . 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 . Finally, recent studies have evaluated the efficacy of autologous ASCs in a rat model of cerebral ischemia . When ASCs were induced to undergo neuronal differentiation upon transplantion, the rats displayed improved neurological recovery and reduced infarct size compared with the controls .
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. . 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 . 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 . 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 . More importantly, ASCs derived hepatocyte-like cells, after transplantation, were able to incorporate into the liver parenchyma of recipient mice . Liver injury repair may also be possible with transplantation of rat ASCs, decreasing key liver enzyme levels and increasing serum albumin . A yet controversial topic is the existence of hepatic stem cells discussed in a recent review .
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. . 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 . 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 . 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 .
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 . 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 . 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 . 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 .
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 . 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 .
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 . Corneal epithelial healing progression was started 11 days after topical application of autologous ASCs. One month later, a complete corneal epithelial healing was observed .
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 .
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 , several clinical trials are been passed on to patients around the world . 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 . 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 . 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 . 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  (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 . 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 .
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 . 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 . 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).