Development of membrane ion channels during neural differentiation from human embryonic stem cells.

OBJECTIVE: For human embryonic stem cells (hESCs) to differentiate into neurons, enormous changes has to occur leading to trigger action potential and neurotransmitter release. We attempt to determine the changes in expression of voltage gated channels (VGCs) and their electrophysiological properties during neural differentiation. MATERIALS AND METHODS: The relative expressions of α-subunit of voltage gated potassium, sodium and calcium channels were characterized by qRT-PCR technique. Patch clamp recording was performed to characterize the electrophysiological properties of hESCs during their differentiation into neuron-like cells. RESULTS: Relative expression of α-subunit of channels changed significantly. 4-AP and TEA sensitive outward currents were observed in all stages, although TEA sensitive currents were recorded once in rosette structure. Nifedipine and QX314 sensitive inward currents were recorded only in neuron-like cells. CONCLUSION: K+ currents were recorded in hESCs and rosette structure cells. Inward currents, sensitive to Nifedipine and QX314, were recorded in neuron-like cells.

Comparative analysis of neural differentiation potential in human mesenchymal stem cells derived from chorion and adult bone marrow.

The finding of a reliable and abundant source of stem cells for the replacement of missing neurons in nervous system diseases requires extensive characterization of neural-differentiation-associated markers in stem cells from various sources. Chorion-derived stem cells from the human placenta have recently been described as an abundant, ethically acceptable, and easily accessible source of cells that are not limited in the same way as bone marrow (BM) mesenchymal stem cells (MSCs). We have isolated and cultured chorion MSCs (C-MSCs) and compared their proliferative capacity, multipotency, and neural differentiation ability with BM-MSCs. C-MSCs showed a higher proliferative capacity compared with BM-MSCs. The expression and histone modification of Nestin, as a marker for neural stem/progenitor cells, was evaluated quantitatively between the two groups. The Nestin expression level in C-MSCs was significantly higher than that in BM-MSCs. Notably, modifications of lys9, lys4, and lys27 of histone H3 agreed with the remarkable higher expression of Nestin in C-MSCs than in BM-MSCs. Furthermore, after neural differentiation of MSCs upon retinoic acid induction, both immunocytochemical and flow cytometry analyses demonstrated that the expression of neural marker genes was significantly higher in neural-induced C-MSCs compared with BM-MSCs. Mature neuron marker genes were also expressed at a significantly higher level in C-MSCs than in BM-MSCs. Thus, C-MSCs have a greater potential than BM-MSCs for differentiation to neural cell lineages and can be regarded as a promising source of stem cells for the cell therapy of neurological disorders.

Astrocytes originated from neural stem cells drive the regenerative remodeling of pathologic CSPGs in spinal cord injury.

Neural degeneration is a hallmark of spinal cord injury (SCI). Multipotent neural precursor cells (NPCs) have the potential to reconstruct the damaged neuron-glia network due to their tri-lineage capacity to generate neurons, astrocytes, and oligodendrocytes. However, astrogenesis is the predominant fate of resident or transplanted NPCs in the SCI milieu adding to the abundant number of resident astrocytes in the lesion. How NPC-derived astrocytes respond to the inflammatory milieu of SCI and the mechanisms by which they contribute to the post-injury recovery processes remain largely unknown. Here, we uncover that activated NPC-derived astrocytes exhibit distinct molecular signature that is immune modulatory and foster neurogenesis, neuronal maturity, and synaptogenesis. Mechanistically, NPC-derived astrocytes perform regenerative matrix remodeling by clearing inhibitory chondroitin sulfate proteoglycans (CSPGs) from the injury milieu through LAR and PTP-σ receptor-mediated endocytosis and the production of ADAMTS1 and ADAMTS9, while most resident astrocytes are pro-inflammatory and contribute to the pathologic deposition of CSPGs. These novel findings unravel critical mechanisms of NPC-mediated astrogenesis in SCI repair.

Controlled Differentiation of Human Neural Progenitor Cells on Molybdenum Disulfide/Graphene Oxide Heterojunction Scaffolds by Photostimulation.

Ultrathin MoS2-MoO3-x heterojunction nanosheets with unique features were introduced as biocompatible, non-cytotoxic, and visible light-sensitive stimulator layers for the controlled differentiation of human neural progenitor cells (hNPCs) into nervous lineages. hNPC differentiation was also investigated on reduced graphene oxide (rGO)-containing scaffolds, that is, rGO and rGO/MoS2-MoO3-x nanosheets. In darkness, hNPC differentiation into neurons increased on MoS2-MoO3-x by a factor of 2.7 due to the excellent biophysical cues and further increased on rGO/MoS2-MoO3-x by a factor of 4.4 due to a synergistic effect induced by the rGO. The MoO3-x domains with antioxidant activity and LSPR absorption induced p-type doping in MoS2-MoO3-x. Under photostimulation, the hNPCs on the MoS2-MoO3-x exhibited higher differentiation into glial cells by a factor of 1.4, and the decrease in photo-electron current to hNPCs due to the induction of more p-type doping in the MoS2-MoO3-x. While the increase in neuronal differentiation of hNPCs on rGO/MoS2-MoO3-x by a factor of 1.8 was ascribed to the presence of rGO as an ultrafast electron transferor which quickly transferred photogenerated electrons to hNPCs before their transfer to free radicals, these results demonstrated the promising potential of MoS2-based scaffolds for applying in the controllable repair and/or regeneration of diseases/disorders related to the nervous system.

Y Chromosome Genes May Play Roles in the Development of Neural Rosettes from Human Embryonic Stem Cells.

BACKGROUND: The human Y chromosome harbors genes that are mainly involved in the growth, development, sexual dimorphism, and spermatogenesis process. Despite many studies, the function of the male-specific region of the Y chromosome (MSY) awaits further clarification, and a cell-based approach can help in this regard. RESULTS: In this study, we have developed four stable transgenic male embryonic stem cell (ESCs) lines that can overexpress male-specific genes HSFY1, RBMY1A1, RPS4Y1, and SRY. As a proof of principle, we differentiated one of these cell lines (RPS4Y1 over-expressing ESCs) to the neural stem cell (rosette structure) and characterized them based on the expression level of lineage markers. RPS4Y1 expression in the Doxycycline-treated group was significantly higher than control groups at transcript and protein levels. Furthermore, we found Doxycycline-treated group had a higher differentiation efficiency than the untreated control groups. CONCLUSIONS: Our results suggest that the RPS4Y1 gene may play a critical role in neurogenesis. Also, the generated transgenic ESC lines can be widely employed in basic and preclinical studies, such as sexual dimorphism of neural and cardiac functions, the development of cancerous and non-cancerous disease models, and drug screening.

Scalable Expansion of Human Pluripotent Stem Cell-Derived Neural Progenitors in Stirred Suspension Bioreactor Under Xeno-free Condition.

Recent advances in neural differentiation technology have paved the way to generate clinical grade neural progenitor populations from human pluripotent stem cells. These cells are an excellent source for the production of neural cell-based therapeutic products to treat incurable central nervous system disorders such as Parkinson's disease and spinal cord injuries. This progress can be complemented by the development of robust bioprocessing technologies for large scale expansion of clinical grade neural progenitors under GMP conditions for promising clinical use and drug discovery applications. Here, we describe a protocol for a robust, scalable expansion of human neural progenitor cells from pluripotent stem cells as 3D aggregates in a stirred suspension bioreactor. The use of this platform has resulted in easily expansion of neural progenitor cells for several passages with a fold increase of up to 4.2 over a period of 5 days compared to a maximum 1.5-2-fold increase in the adherent static culture over a 1 week period. In the bioreactor culture, these cells maintained self-renewal, karyotype stability, and cloning efficiency capabilities. This approach can be also used for human neural progenitor cells derived from other sources such as the human fetal brain.

Conversion of Human Fibroblasts to Stably Self-Renewing Neural Stem Cells with a Single Zinc-Finger Transcription Factor.

Direct conversion of somatic cells into neural stem cells (NSCs) by defined factors holds great promise for mechanistic studies, drug screening, and potential cell therapies for different neurodegenerative diseases. Here, we report that a single zinc-finger transcription factor, Zfp521, is sufficient for direct conversion of human fibroblasts into long-term self-renewable and multipotent NSCs. In vitro, Zfp521-induced NSCs maintained their characteristics in the absence of exogenous factor expression and exhibited morphological, molecular, developmental, and functional properties that were similar to control NSCs. In addition, the single-seeded induced NSCs were able to form NSC colonies with efficiency comparable with control NSCs and expressed NSC markers. The converted cells were capable of surviving, migrating, and attaining neural phenotypes after transplantation into neonatal mouse and adult rat brains, without forming tumors. Moreover, the Zfp521-induced NSCs predominantly expressed rostral genes. Our results suggest a facilitated approach for establishing human NSCs through Zfp521-driven conversion of fibroblasts.

Behaviour of human induced pluripotent stem cell-derived neural progenitors on collagen scaffolds varied in freezing temperature and laminin concentration.

OBJECTIVE: Biomaterial technology, when combined with emerging human induced pluripotent stem cell (hiPSC) technology, provides a promising strategy for patient-specific tissue engineering. In this study, we have evaluated the physical effects of collagen scaffolds fabricated at various freezing temperatures on the behavior of hiPSC-derived neural progenitors (hiPSC-NPs). In addition, the coating of scaffolds using different concentrations of laminin was examined on the cells. MATERIALS AND METHODS: Initially, in this experimental study, the collagen scaffolds fabricated from different collagen concentrations and freezing temperatures were characterized by determining the pore size, porosity, swelling ratio, and mechanical properties. Effects of cross-linking on free amine groups, volume shrinkage and mass retention was also assessed. Then, hiPSC-NPs were seeded onto the most stable three-dimensional collagen scaffolds and we evaluated the effect of pore structure. Additionally, the different concentrations of laminin coating of the scaffolds on hiPSC-NPs behavior were assessed. RESULTS: Scanning electron micrographs of the scaffolds showed a pore diameter in the range of 23-232 µm for the scaffolds prepared with different fabrication parameters. Also porosity of all scaffolds was >98% with more than 94% swelling ratio. hiPSC-NPs were subsequently seeded onto the scaffolds that were made by different freezing temperatures in order to assess for physical effects of the scaffolds. We observed similar proliferation, but more cell infiltration in scaffolds prepared at lower freezing temperatures. The laminin coating of the scaffolds improved NPs proliferation and infiltration in a dose-dependent manner. Immunofluorescence staining and scanning electron microscopy confirmed the compatibility of undifferentiated and differentiated hiPSC-NPs on these scaffolds. CONCLUSION: The results have suggested that the pore structure and laminin coating of collagen scaffolds significantly impact cell behavior. These biocompatible three-dimensional laminin-coated collagen scaffolds are good candidates for future hiPSC-NPs biomedical nerve tissue engineering applications.

Transplantation of adult monkey neural stem cells into a contusion spinal cord injury model in rhesus macaque monkeys.

OBJECTIVE: Currently, cellular transplantation for spinal cord injuries (SCI) is the subject of numerous preclinical studies. Among the many cell types in the adult brain, there is a unique subpopulation of neural stem cells (NSC) that can self-renew and differentiate into neurons. The study aims, therefore, to explore the efficacy of adult monkey NSC (mNSC) in a primate SCI model. MATERIALS AND METHODS: In this experimental study, isolated mNSCs were analyzed by flow cytometry, immunocytochemistry, and RT-PCR. Next, BrdU-labeled cells were transplanted into a SCI model. The SCI animal model was confirmed by magnetic resonance imaging (MRI) and histological analysis. Animals were clinically observed for 6 months. RESULTS: Analysis confirmed homing of mNSCs into the injury site. Transplanted cells expressed neuronal markers (TubIII). Hind limb performance improved in trans- planted animals based on Tarlov's scale and our established behavioral tests for monkeys. CONCLUSION: Our findings have indicated that mNSCs can facilitate recovery in contusion SCI models in rhesus macaque monkeys. Additional studies are necessary to determine the im- provement mechanisms after cell transplantation.

Cloning, expression, and functional characterization of in-house prepared human leukemia inhibitory factor.

OBJECTIVE: Leukemia inhibitory factor (LIF) plays important roles in cellular proliferation, growth promotion and differentiation of various types of target cells. In addition, LIF influences bone metabolism, cachexia, neural development, embryogenesis and inflammation. Human LIF (hLIF) is an essential growth factor for the maintenance of mouse embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) in a pluripotent, undifferentiated state. MATERIALS AND METHODS: In this experimental study, we cloned hLIF into the pENTR-D/ TOPO entry vector by a TOPO reaction. Next, hLIF was subcloned into the pDEST17 destination vector by the LR reaction, which resulted in the production of a construct that was transferred into E. coli strain Rosetta-gami™ 2(DE3) pLacI competent cells to produce the His6-hLIF fusion protein. RESULTS: This straightforward method produced a biologically active recombinant hLIF protein in E. coli that has long-term storage ability. This procedure has provided rapid, cost effective purification of a soluble hLIF protein that is biologically active and functional as measured in mouse ESCs and iPSCs in vitro. CONCLUSION: Our results showed no significant differences in function between laboratory produced and commercialized hLIF.

Glycogen synthase kinase-3 inhibition promotes proliferation and neuronal differentiation of human-induced pluripotent stem cell-derived neural progenitors.

Human-induced pluripotent stem cell-derived neural progenitors (hiPSC-NPs) have the ability to self-renew and differentiate into glial and neuronal lineages, which makes them an invaluable source in cell replacement therapy for neurological diseases. Therefore, their enhanced proliferation and neuronal differentiation are pivotal features that can be used in repairing neurological injuries. One of the main regulators of neural development is Wnt signaling, which results in the inhibition of glycogen synthase kinase 3 (GSK-3). Here, we assess the impact of GSK-3 inhibition by the small molecule CHIR99021 on the expansion and differentiation of hiPSC-NPs in an adherent condition and a defined medium. Cell proliferation analyses have revealed that inhibition of GSK-3 in the presence of epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) increased the proliferation of hiPSC-NPs across 10 passages. The inhibition of ß-catenin signaling by XAV and NOTCH signaling by DAPT reversed CHIR impact on hiPSC-NPs proliferation. The target genes of ß-catenin, C-MYC and CYCLIN D1 as well as NOTCH target genes, HES1 and HES5 were upregulated. The treatment of NPs by CHIR in the absence of bFGF and EGF resulted in an increase of neuronal differentiation rather than proliferation by stabilization of ß-catenin regardless of the NOTCH pathway. Thus, GSK-3 inhibition has been shown to promote proliferation of the NPs by activating ß-catenin and NOTCH-related cell cycle genes in the presence of bFGF and EGF. Additionally, during GSK-3 inhibition, an absence of these growth factors allows for the switch to neuronal differentiation with a bias toward a dopaminergic fate. This may provide desired cells that can be used in therapeutic applications and offer insights into the etiology of some neurological disorders.

Cotransplantation of human embryonic stem cell-derived neural progenitors and schwann cells in a rat spinal cord contusion injury model elicits a distinct neurogenesis and functional recovery.

Cotransplantation of neural progenitors (NPs) with Schwann cells (SCs) might be a way to overcome low rate of neuronal differentiation of NPs following transplantation in spinal cord injury (SCI) and the improvement of locomotor recovery. In this study, we initially generated NPs from human embryonic stem cells (hESCs) and investigated their potential for neuronal differentiation and functional recovery when cocultured with SCs in vitro and cotransplanted in a rat acute model of contused SCI. Cocultivation results revealed that the presence of SCs provided a consistent status for hESC-NPs and recharged their neural differentiation toward a predominantly neuronal fate. Following transplantation, a significant functional recovery was observed in all engrafted groups (NPs, SCs, NPs + SCs) relative to the vehicle and control groups. We also observed that animals receiving cotransplants established a better state as assessed with the BBB functional test. Immunohistofluorescence evaluation 5 weeks after transplantation showed invigorated neuronal differentiation and limited proliferation in the cotransplanted group when compared to the individual hESC-NP-grafted group. These findings have demonstrated that the cotransplantation of SCs with hESC-NPs could offer a synergistic effect, promoting neuronal differentiation and functional recovery.

Long-term self-renewable feeder-free human induced pluripotent stem cell-derived neural progenitors.

Human induced pluripotent stem cells (hiPSCs) have led to an important revolution in stem cell research and regenerative medicine. To create patient-specific neural progenitors (NPs), we have established a homogenous, expandable, and self-renewable population of multipotent NPs from hiPSCs, using an adherent system and defined medium supplemented with a combination of factors. The established hiPSC-NPs highly expressed Nestin and Sox1. These NPs were continuously propagated for ~1 year without losing their potential to generate astrocytes, oligodendrocytes, and functional neurons and maintained a stable chromosome number. Voltage clamp analysis revealed outward potassium currents in hiPSC-NPs. The self-renewal characteristic of the NPs was demonstrated by a symmetrical mode of Nestin-positive cell division. Additionally, these hiPSC-NPs can be easily frozen and thawed in the presence of Rho-associated kinase (ROCK) inhibitor without losing their proliferation, karyotype stability, and developmental potential. The characteristics of our generated hiPSC-NPs provide the opportunity to use patient-specific or ready-to-use hiPSC-NPs in future biomedical applications.