Masaaki Kitada

Specific induction of neuronal cells from bone marrow stromal cells and application for autologous transplantation

Bone marrow stromal cells (MSCs) have the capability under specific conditions of differentiating into various cell types such as osteocytes, chondrocytes, and adipocytes. Here we demonstrate a highly efficient and specific induction of cells with neuronal characteristics, without glial differentiation, from both rat and human MSCs using gene transfection with Notch intracellular domain (NICD) and subsequent treatment with bFGF, forskolin, and ciliary neurotrophic factor. MSCs expressed markers related to neural stem cells after transfection with NICD, and subsequent trophic factor administration induced neuronal cells. Some of them showed voltage-gated fast sodium and delayed rectifier potassium currents and action potentials compatible with characteristics of functional neurons. Further treatment of the induced neuronal cells with glial cell line-derived neurotrophic factor (GDNF) increased the proportion of tyrosine hydroxylase-positive and dopamine-producing cells. Transplantation of these GDNF-treated cells showed improvement in apomorphine-induced rotational behavior and adjusting step and paw-reaching tests following intrastriatal implantation in a 6-hydroxy dopamine rat model of Parkinson disease. This study shows that a population of neuronal cells can be specifically generated from MSCs and that induced cells may allow for a neuroreconstructive approach.

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.

Bone marrow stromal cells enhance differentiation of cocultured neurosphere cells and promote regeneration of injured spinal cord

Transplantation of bone marrow stromal cells (MSCs) has been regarded as a potential approach for promoting nerve regeneration. In the present study, we investigated the influence of MSCs on spinal cord neurosphere cells in vitro and on the regeneration of injured spinal cord in vivo by grafting. MSCs from adult rats were cocultured with fetal spinal cord‐derived neurosphere cells by either cell mixing or making monolayered‐feeder cultures. In the mixed cell cultures, neuroshpere cells were stimulated to develop extensive processes. In the monolayered‐feeder cultures, numerous processes from neurosphere cells appeared to be attracted to MSCs. In an in vivo experiment, grafted MSCs promoted the regeneration of injured spinal cord by enhancing tissue repair of the lesion, leaving apparently smaller cavities than in controls. Although the number of grafted MSCs gradually decreased, some treated animals showed remarkable functional recovery. These results suggest that MSCs might have profound effects on the differentiation of neurosphere cells and be able to promote regeneration of the spinal cord by means of grafting.

Development of NG2 neural progenitor cells requires Olig gene function

In the adult central nervous system, two distinct populations of glial cells expressing the chondroitin sulfate proteoglycan NG2 have been described: bipolar progenitor cells and more differentiated “synantocytes.” These cells have diverse neurological functions, including critical roles in synaptic transmission, repair, and regeneration. Despite their potential importance, the genetic factors that regulate NG2 cell development are poorly understood, and the relationship of synantocytes to the oligodendroglial lineage, in particular, remains controversial. Here, we show that >90% of embryonic and adult NG2 cells express Olig2, a basic helix–loop–helix transcription factor required for oligodendrocyte lineage specification. Analysis of mice lacking Olig function demonstrates a failure of NG2 cell development at embryonic and perinatal stages that can be rescued by addition of a transgene containing the human OLIG2 locus. These findings show a general requirement for Olig function in NG2 cell development and highlight further roles for Olig transcription factors in neural progenitor cells.

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.

Alginate Enhances Elongation of Early Regenerating Axons in Spinal Cord of Young Rats

Freeze-dried alginate sponge cross-linked with covalent bonds has been demonstrated to enhance nerve regeneration in peripheral nerves and spinal cords. The present study examined, at early stages after surgery, the outgrowth of regenerating axons and reactions of astrocytes at the stump of transected spinal cord in young rats. Two segments (Th7-8) were resected, and alginate was implanted in the lesion. As controls, collagen gel was implanted in place of alginate or the lesion was left without implantation. Two and 4 weeks after surgery, nerve outgrowth and astrocyte reactions were examined. Many regenerating axons, some of which were accompanied by astrocytic processes, were found to extend from the stump into the alginate-implanted lesion. In the all nonimplanted animals, large cystic cavities were formed at both interfaces with no definite axonal outgrowth into the lesion. In collagen-implanted animals, cavity formation was found in some rats, and regenerating axons once formed at the stumps did not extend further into the lesion. Astrocytic processes extending into alginate-implanted lesion had no basal laminae, whereas those found in control experiments were covered by basal laminae. These findings suggest that alginate contributed to reducing the barrier composed of connective tissues and reactive astrocytic processes, and served as a scaffold for the outgrowth of regenerating axons and elongation of astrocytic processes.

Migration, integration, and differentiation of hippocampus-derived neurosphere cells after transplantation into injured rat spinal cord

Hippocampus-derived neurospheres were prepared from transgenic rat fetuses expressing green fluorescent protein (GFP), and transplanted into an alginate-filled lesion of young rat spinal cord. One, two and four weeks after transplantation, a large number of grafted cells survived, many of which expressed immunoreactivity for glial fibrillary acidic protein, and a few expressed immunoreactivity for beta-tubulin III. The grafted cells closely attached to the host tissue including astrocytes at the border of the lesion. It was notable that numerous GFP-positive cells had migrated within host spinal cord tissue up to 2 mm away from the implanted site 4 weeks postoperation. These results demonstrate that rat fetal hippocampus-derived neurosphere cells could survive, differentiate, extensively migrate, and integrate well into the host spinal cord tissue.

Alginate, a bioresorbable material derived from brown seaweed, enhances elongation of amputated axons of spinal cord in infant rats

Freeze-dried alginate sponge crosslinked with covalent bonds was developed in our laboratory and has been demonstrated to enhance peripheral nerve regeneration. In this study, we examined spinal cord repair using alginate sponge in infant rats. On postnatal day 8-12, the spinal cord was transversely resected at Th7-Th8 to produce a 2-mm gap. The gap was filled with alginate sponge in the alginate group. For the control group, the gap was left empty. In the alginate group, the recovery of evoked electromyogram and sensory-evoked potentials 6 weeks after surgery indicated that elongation of axons could establish electrophysiologically functional projections through the gap. A histological study revealed that myelinated and unmyelinated axons, surrounded by a perineurial-like structure, had elongated across the gap. An immunohistochemical examination revealed that elongation of astrocytic processes and/or migration of astrocytes into the alginate sponge was induced, whereas astrocyte gliosis was reduced at the interface between the implanted alginate and the host spinal cord, compared with the control group. However, a horseradish peroxidase tracing study revealed ascending and descending fibers had also elongated into the gap and reentered the other stump of the transected spinal cord beyond the gap. These results suggest that alginate might provide a permissive microenvironment for elongation of spinal cord axons.

Committed neural progenitor cells derived from genetically modified bone marrow stromal cells ameliorate deficits in a rat model of stroke

Bone marrow stromal cells (MSCs) are an excellent source of cells for treating a variety of central nervous system diseases. In this study, we report the efficient induction of committed neural progenitor cells from rat and human MSCs (NS-MSCs) by introduction of cells with the intracellular domain of Notch-1 followed by growth in the free-floating culture system. NS-MSCs successfully formed spheres, in which cells highly expressed the neural precursor cell markers. The commitment of spheres to neural lineage cells was confirmed by their successful differentiation into neuronal cells when exposed to a differentiation medium. To determine the therapeutic potential of NS-MSCs, cells were transplanted into the cortex and striatum in a rat model of focal cerebral ischemia. The survival, distribution, and integration of NS-MSCs in the host brain were very high, and at day 100, grafted NS-MSCs were positive for dopaminergic, glutamatergic, and γ-amino butyric acid (GABA)ergic neuronal markers. They extended long neurites for nearly 6.3 mm and many of these expressed synaptophysin. Significant behavioral recovery was also observed in limb-placing and water-maze tests. These suggest a high potential for this MSC approach in the replenishment of neural cells for stroke and for a wide range of neurodegenerative conditions that require various types of neural cells.

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.