Cortical stem cell transplantation may help replace lost brain cells after stroke and improve the functional outcome. In this study, we transplanted human embryonic stem cell (hESC)-derived neural precursor cells (hNPCs) or vehicle into the cortex of rats after permanent distal middle cerebral artery occlusion (dMCAO) or sham-operation, and followed functional recovery in the cylinder and staircase tests. The hNPCs were examined prior to transplantation, and they expressed neuroectodermal markers but not markers for undifferentiated hESCs or non-neural cells. The rats were housed in either enriched environment or standard cages to examine the effects of additive rehabilitative therapy. In the behavioral tests dMCAO groups showed significant impairments compared with sham group before transplantation. Vehicle groups remained significantly impaired in the cylinder test 1 and 2 months after vehicle injection, whereas hNPC transplanted groups did not differ from the sham group. Rehabilitation or hNPC transplantation had no effect on reaching ability measured in the staircase test, and no differences were found in the cortical infarct volumes. After 2 months we measured cell survival and differentiation in vivo using stereology and confocal microscopy. Housing had no effect on cell survival or differentiation. The majority of the transplanted hNPCs were positive for the neural precursor marker nestin. A portion of transplanted cells expressed neuronal markers 2 months after transplantation, whereas only a few cells co-localized with astroglial or oligodendrocyte markers. In conclusion, hESC-derived neural precursor transplants provided some improvement in sensorimotor function after dMCAO, but did not restore more complicated sensorimotor functions.
The great potential of human embryonic stem (hES) cells offers the opportunity both for studying basic developmental processes in vitro as well as for drug screening, modeling diseases, or future cell therapy. Defining protocols for the generation of human neural progenies represents a most important prerequisite. Here, we have used six hES cell lines to evaluate defined conditions for neural differentiation in suspension and adherent culture systems. Our protocol does not require fetal serum, feeder cells, or retinoic acid at any step, to induce neural fate decisions in hES cells. We monitored neurogenesis in differentiating cultures using morphological (including on-line follow up), immunocytochemical, and RT-PCR assays. For each hES cell line, in suspension or adherent culture, the same longitudinal progression of neural differentiation occurs. We showed the dynamic transitions from hES cells to neuroepithelial (NE) cells, to radial glial (RG) cells, and to neurons. Thus, 7 days after neural induction the majority of cells were NE, expressing nestin, Sox1, and Pax6. During neural proliferation and differentiation, NE cells transformed in RG cells, which acquired vimentin, BLBP, GLAST, and GFAP, proliferated and formed radial scaffolds. gamma-Aminobutyric acid (GABA)-positive and glutamate positive neurons, few oligodendrocyte progenitors and astrocytes were formed in our conditions and timing. Our system successfully generates human RG cells and could be an effective source for neuronal replacement, since RG cells predominantly generate neurons and provide them with support and guidance.
BackgroundThe growth of stem cells in in vitro conditions requires optimal balance between signals mediating cell survival, proliferation, and self-renewal. For clinical application of stem cells, the use of completely defined conditions and elimination of all animal-derived materials from the establishment, culture, and differentiation processes is desirable.Methodology/Principal FindingsHere, we report the development of a fully defined xeno-free medium (RegES), capable of supporting the expansion of human embryonic stem cells (hESC), induced pluripotent stem cells (iPSC) and adipose stem cells (ASC). We describe the use of the xeno-free medium in the derivation and long-term (>80 passages) culture of three pluripotent karyotypically normal hESC lines: Regea 06/015, Regea 07/046, and Regea 08/013. Cardiomyocytes and neural cells differentiated from these cells exhibit features characteristic to these cell types. The same formulation of the xeno-free medium is capable of supporting the undifferentiated growth of iPSCs on human feeder cells. The characteristics of the pluripotent hESC and iPSC lines are comparable to lines derived and cultured in standard undefined culture conditions. In the culture of ASCs, the xeno-free medium provided significantly higher proliferation rates than ASCs cultured in medium containing allogeneic human serum (HS), while maintaining the differentiation potential and characteristic surface marker expression profile of ASCs, although significant differences in the surface marker expression of ASCs cultured in HS and RegES media were revealed.Conclusion/SignificanceOur results demonstrate that human ESCs, iPSCs and ASCs can be maintained in the same defined xeno-free medium formulation for a prolonged period of time while maintaining their characteristics, demonstrating the applicability of the simplified xeno-free medium formulation for the production of clinical-grade stem cells. The basic xeno-free formulation described herein has the potential to be further optimized for specific applications relating to establishment, expansion and differentiation of various stem cell types.
Human pluripotent stem cell (hPSC)-derived neurons provide exciting opportunities for in vitro modeling of neurological diseases and for advancing drug development and neurotoxicological studies. However, generating electrophysiologically mature neuronal networks from hPSCs has been challenging. Here, we report the differentiation of functionally active hPSC-derived cortical networks on defined laminin-521 substrate. We apply microelectrode array (MEA) measurements to assess network events and compare the activity development of hPSC-derived networks to that of widely used rat embryonic cortical cultures. In both of these networks, activity developed through a similar sequence of stages and time frames; however, the hPSC-derived networks showed unique patterns of bursting activity. The hPSC-derived networks developed synchronous activity, which involved glutamatergic and GABAergic inputs, recapitulating the classical cortical activity also observed in rodent counterparts. Principal component analysis (PCA) based on spike rates, network synchronization and burst features revealed the segregation of hPSC-derived and rat network recordings into different clusters, reflecting the species-specific and maturation state differences between the two networks. Overall, hPSC-derived neural cultures produced with a defined protocol generate cortical type network activity, which validates their applicability as a human-specific model for pharmacological studies and modeling network dysfunctions.
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