Shohei Wakao

Unique multipotent cells in adult human mesenchymal cell populations

We found adult human stem cells that can generate, from a single cell, cells with the characteristics of the three germ layers. The cells are stress-tolerant and can be isolated from cultured skin fibroblasts or bone marrow stromal cells, or directly from bone marrow aspirates. These cells can self-renew; form characteristic cell clusters in suspension culture that express a set of genes associated with pluripotency; and can differentiate into endodermal, ectodermal, and mesodermal cells both in vitro and in vivo. When transplanted into immunodeficient mice by local or i.v. injection, the cells integrated into damaged skin, muscle, or liver and differentiated into cytokeratin 14-, dystrophin-, or albumin-positive cells in the respective tissues. Furthermore, they can be efficiently isolated as SSEA-3(+) cells. Unlike authentic ES cells, their proliferation activity is not very high and they do not form teratomas in immunodeficient mouse testes. Thus, nontumorigenic stem cells with the ability to generate the multiple cell types of the three germ layers can be obtained through easily accessible adult human mesenchymal cells without introducing exogenous genes. These unique cells will be beneficial for cell-based therapy and biomedical research.

Multilineage-differentiating stress-enduring (Muse) cells are a primary source of induced pluripotent stem cells in human fibroblasts

The stochastic and elite models have been proposed for the mechanism of induced pluripotent stem (iPS) cell generation. In this study we report a system that supports the elite model. We previously identified multilineage-differentiating stress-enduring (Muse) cells in human dermal fibroblasts that are characterized by stress tolerance, expression of pluripotency markers, self-renewal, and the ability to differentiate into endodermal-, mesodermal-, and ectodermal-lineage cells from a single cell. They can be isolated as stage-specific embryonic antigen-3/CD105 double-positive cells. When human fibroblasts were separated into Muse and non-Muse cells and transduced with Oct3/4, Sox2, Klf4, and c-Myc, iPS cells were generated exclusively from Muse cells but not from non-Muse cells. Although some colonies were formed from non-Muse cells, they were unlike iPS cells. Furthermore, epigenetic alterations were not seen, and some of the major pluripotency markers were not expressed for the entire period during iPS cell generation. These findings were confirmed further using cells transduced with a single polycistronic virus vector encoding all four factors. The results demonstrate that in adult human fibroblasts a subset of preexisting adult stem cells whose properties are similar in some respects to those of iPS cells selectively become iPS cells, but the remaining cells make no contribution to the generation of iPS cells. Therefore this system seems to fit the elite model rather than the stochastic model.

Isolation, culture and evaluation of multilineage-differentiating stress-enduring (Muse) cells

Multilineage-differentiating stress-enduring (Muse) cells are distinct stem cells in mesenchymal cell populations with the capacity to self-renew, to differentiate into cells representative of all three germ layers from a single cell, and to repair damaged tissues by spontaneous differentiation into tissue-specific cells without forming teratomas. We describe step-by-step procedures for isolating and evaluating these cells. Muse cells are also a practical cell source for human induced pluripotent stem (iPS) cells with markedly high generation efficiency. They can be collected as cells that are double positive for stage-specific embryonic antigen-3 (SSEA-3) and CD105 from commercially available mesenchymal cells, such as adult human bone marrow stromal cells and dermal fibroblasts, or from fresh adult human bone marrow samples. Under both spontaneous and induced differentiation conditions, they show triploblastic differentiation. It takes 4–6 h to collect and 2 weeks to confirm the differentiation and self-renewal capacity of Muse cells.

Long-term observation of auto-cell transplantation in non-human primate reveals safety and efficiency of bone marrow stromal cell-derived Schwann cells in peripheral nerve regeneration

Based on their differentiation ability, bone marrow stromal cells (MSCs) are a good source for cell therapy. Using a cynomolgus monkey peripheral nervous system injury model, we examined the safety and efficacy of Schwann cells induced from MSCs as a source for auto-cell transplantation therapy in nerve injury. Serial treatment of monkey MSCs with reducing agents and cytokines induced their differentiation into cells with Schwann cell properties at a very high ratio. Expression of Schwann cell markers was confirmed by both immunocytochemistry and reverse transcription-polymerase chain reaction. Induced Schwann cells were used for auto-cell transplantation into the median nerve and followed-up for 1year. No abnormalities were observed in general conditions. Ki67-immunostaining revealed no sign of massive proliferation inside the grafted tube. Furthermore, (18)F-fluorodeoxygluocose-positron emission tomography scanning demonstrated no abnormal accumulation of radioactivity except in regions with expected physiologic accumulation. Restoration of the transplanted nerve was corroborated by behavior analysis, electrophysiology and histological evaluation. Our results suggest that auto-cell transplantation therapy using MSC-derived Schwann cells is safe and effective for accelerating the regeneration of transected axons and for functional recovery of injured nerves. The practical advantages of MSCs are expected to make this system applicable for spinal cord injury and other neurotrauma or myelin disorders where the acceleration of regeneration is expected to enhance functional recovery.

Human umbilical cord-derived mesenchymal stromal cells differentiate into functional schwann cells that sustain peripheral nerve regeneration

Human umbilical cord-derived mesenchymal stromal cells (UC-MSCs) that are available from cell banks can be induced to differentiate into various cell types, thereby making them practical potential sources for cell-based therapies. In injured peripheral nerves, Schwann cells (SCs) contribute to functional recovery by supporting axonal regeneration and myelin reconstruction. Here, we first demonstrate a system toinduce UC-MSCs to differentiate into cells with SC properties (UC-SCs) by treatment with &bgr;-mercaptoethanol followed by retinoic acid and a set of specific cytokines. The UC-SCs are morphologically similar to SCs and express SC markers, including P0, as assessed by immunocytochemistry and reverse transcription polymerase chain reaction. Transplantation of UC-SCs into transected sciatic nerves in adult rats enhanced nerve regeneration. The effectiveness of UC-SCs for axonal regeneration was comparable to that of authentic human SCs based on histological criteria and functional recovery. Immunohistochemistry and immunoelectron microscopy also demonstrated myelination of regenerated axons by UC-SCs. These findings indicate that cells with SC properties and with the ability to support axonal regeneration and reconstruct myelin can be successfully induced from UC-MSCs to promote functional recovery after peripheral nerve injury. This system may be applicable for the development of cell-based therapies.

Bone Marrow Mesenchymal Cells: How Do They Contribute to Tissue Repair and Are They Really Stem Cells?

Adult stem cells typically generate the cell types of the tissue in which they reside, and thus the range of their differentiation is considered limited. Bone marrow mesenchymal stem cells (MSCs) are different from other somatic stem cells in that they differentiate not only into the same mesodermal-lineage such as bone, cartilage, and adipocytes but also into other lineages of ectodermal and endodermal cells. Thus, MSCs are a unique type of adult stem cells. In addition, MSCs home to damaged sites, differentiate into cells specific to the tissue and contribute to tissue repair. Therefore, application of MSCs in the treatment of various diseases, including liver dysfunction, myocardial infarction, and central nervous system repair, has been initiated. Because MSCs are generally harvested as adherent cells from bone marrow aspirates, however, they comprise heterogeneous cell populations and their wide-ranging differentiation ability and repair functions are not yet clear. Recent evidence suggests that a very small subpopulation of cells that assume a repair function with the ability to differentiate into trilineage cells resides among human MSCs and effective utilization of such cells is expected to improve the repair effect of MSCs. This review summarizes recent advances in the clarification of MSC properties and discusses future perspectives.

Autologous mesenchymal stem cell–derived dopaminergic neurons function in parkinsonian macaques

A cell-based therapy for the replacement of dopaminergic neurons has been a long-term goal in Parkinsons disease research. Here, we show that autologous engraftment of A9 dopaminergic neuron-like cells induced from mesenchymal stem cells (MSCs) leads to long-term survival of the cells and restoration of motor function in hemiparkinsonian macaques. Differentiated MSCs expressed markers of A9 dopaminergic neurons and released dopamine after depolarization in vitro. The differentiated autologous cells were engrafted in the affected portion of the striatum. Animals that received transplants showed modest and gradual improvements in motor behaviors. Positron emission tomography (PET) using [11C]-CFT, a ligand for the dopamine transporter (DAT), revealed a dramatic increase in DAT expression, with a subsequent exponential decline over a period of 7 months. Kinetic analysis of the PET findings revealed that DAT expression remained above baseline levels for over 7 months. Immunohistochemical evaluations at 9 months consistently demonstrated the existence of cells positive for DAT and other A9 dopaminergic neuron markers in the engrafted striatum. These data suggest that transplantation of differentiated autologous MSCs may represent a safe and effective cell therapy for Parkinsons disease.

Regenerative Effects of Mesenchymal Stem Cells: Contribution of Muse Cells, a Novel Pluripotent Stem Cell Type that Resides in Mesenchymal Cells

Mesenchymal stem cells (MSCs) are easily accessible and safe for regenerative medicine. MSCs exert trophic, immunomodulatory, anti-apoptotic, and tissue regeneration effects in a variety of tissues and organs, but their entity remains an enigma. Because MSCs are generally harvested from mesenchymal tissues, such as bone marrow, adipose tissue, or umbilical cord as adherent cells, MSCs comprise crude cell populations and are heterogeneous. The specific cells responsible for each effect have not been clarified. The most interesting property of MSCs is that, despite being adult stem cells that belong to the mesenchymal tissue lineage, they are able to differentiate into a broad spectrum of cells beyond the boundary of mesodermal lineage cells into ectodermal or endodermal lineages, and repair tissues. The broad spectrum of differentiation ability and tissue-repairing effects of MSCs might be mediated in part by the presence of a novel pluripotent stem cell type recently found in adult human mesenchymal tissues, termed multilineage-differentiating stress enduring (Muse) cells. Here we review recently updated studies of the regenerative effects of MSCs and discuss their potential in regenerative medicine.

Functional Melanocytes Are Readily Reprogrammable from Multilineage-Differentiating Stress-Enduring (Muse) Cells, Distinct Stem Cells in Human Fibroblasts

The induction of melanocytes from easily accessible stem cells has attracted attention for the treatment of melanocyte dysfunctions. We found that multilineage-differentiating stress-enduring (Muse) cells, a distinct stem cell type among human dermal fibroblasts, can be readily reprogrammed into functional melanocytes, whereas the remainder of the fibroblasts do not contribute to melanocyte differentiation. Muse cells can be isolated as cells positive for stage-specific embryonic antigen-3, a marker for undifferentiated human embryonic stem cells, and differentiate into cells representative of all three germ layers from a single cell, while also being nontumorigenic. The use of certain combinations of factors induces Muse cells to express melanocyte markers such as tyrosinase and microphthalmia-associated transcription factor and to show positivity for the 3,4-dihydroxy-L-phenylalanine reaction. When Muse cell-derived melanocytes were incorporated into three-dimensional (3D) cultured skin models, they localized themselves in the basal layer of the epidermis and produced melanin in the same manner as authentic melanocytes. They also maintained their melanin production even after the 3D cultured skin was transplanted to immunodeficient mice. This technique may be applicable to the efficient production of melanocytes from accessible human fibroblasts by using Muse cells, thereby contributing to autologous transplantation for melanocyte dysfunctions, such as vitiligo.

A Distinct Subpopulation of Bone Marrow Mesenchymal Stem Cells, Muse Cells, Directly Commit to the Replacement of Liver Components.

Genotyping graft livers by short tandem repeats after human living‐donor liver transplantation (n = 20) revealed the presence of recipient or chimeric genotype cases in hepatocytes (6 of 17, 35.3%), sinusoidal cells (18 of 18, 100%), cholangiocytes (15 of 17, 88.2%) and cells in the periportal areas (7 of 8, 87.5%), suggesting extrahepatic cell involvement in liver regeneration. Regarding extrahepatic origin, bone marrow mesenchymal stem cells (BM‐MSCs) have been suggested to contribute to liver regeneration but compose a heterogeneous population. We focused on a more specific subpopulation (1–2% of BM‐MSCs), called multilineage‐differentiating stress‐enduring (Muse) cells, for their ability to differentiate into liver‐lineage cells and repair tissue. We generated a physical partial hepatectomy model in immunodeficient mice and injected green fluorescent protein (GFP)‐labeled human BM‐MSC Muse cells intravenously (n = 20). Immunohistochemistry, fluorescence in situ hybridization and species‐specific polymerase chain reaction revealed that they integrated into regenerating areas and expressed liver progenitor markers during the early phase and then differentiated spontaneously into major liver components, including hepatocytes (≈74.3% of GFP‐positive integrated Muse cells), cholangiocytes (≈17.7%), sinusoidal endothelial cells (≈2.0%), and Kupffer cells (≈6.0%). In contrast, the remaining cells in the BM‐MSCs were not detected in the liver for up to 4 weeks. These results suggest that Muse cells are the predominant population of BM‐MSCs that are capable of replacing major liver components during liver regeneration.