NSC-like Cells Research Articles

A comparative study of the capability of MSCs isolated from different human tissue sources to differentiate into neuronal stem cells and dopaminergic-like cells.

BackgroundNeurodegenerative diseases are characterized by progressive neuronal loss and degeneration. The regeneration of neurons is minimal and neurogenesis is limited only to specific parts of the brain. Several clinical trials have been conducted using Mesenchymal Stem Cells (MSCs) from different sources to establish their safety and efficacy for the treatment of several neurological disorders such as Parkinson’s disease, multiple sclerosis and amyotrophic lateral sclerosis.AimThe aim of this study was to provide a comparative view of the capabilities of MSCs, isolated from different human tissue sources to differentiate into neuronal stem cell-like cells (NSCs) and possibly into dopaminergic neural- like cells.MethodsMesenchymal stem cells were isolated from human bone marrow, adipose, and Wharton’s jelly (WJ) tissue samples. Cells were characterized by flow cytometry for their ability to express the most common MSC markers. The differentiation potential was also assessed by differentiating them into osteogenic and adipogenic cell lineages. To evaluate the capacity of these cells to differentiate towards the neural stem cell-like lineage, cells were cultured in media containing small molecules. Cells were utilized for gene expression and immunofluorescence analysis at different time points.ResultsOur results indicate that we have successfully isolated MSCs from bone marrow, adipose tissue, and Wharton’s jelly. WJ-MSCs showed a slightly higher proliferation rate after 72 hours compared to BM and AT derived MSCs. Gene expression of early neural stem cell markers revealed that WJ-MSCs had higher expression of Nestin and PAX6 compared to BM and AT-MSCs, in addition to LMX expression as an early dopaminergic neural marker. Immunofluorescence analysis also revealed that these cells successfully expressed SOX1, SOX2, Nestin, TUJ1, FOXA2 and TH.ConclusionThese results indicate that the protocol utilized has successfully differentiated BM, AT and WJ-MSCs into NSC-like cells. WJ-MSCs possess a higher potential to transdifferentiate into NSC and dopaminergic-like cells. Thus, it might indicate that this protocol can be used to induce MSC into neuronal lineage, which provides an additional or alternative source of cells to be used in the neurological cell-based therapies.

Sox-positive cell population in the adult cerebellum increases upon tissue degeneration

Adult neurogenesis is well-described in the subventricular and subgranular zones of the mammalian brain. Recent observations that resident glia express stem cell markers in some areas of the brain not traditionally associated with neurogenesis hint to a possible role in tissue repair. The Bergmann glia (BG) population in the cerebellum displays markers and in vitro features associated with neural stem cells (NSC), however the physiological relevance of this phenotypic overlap remains unclear in the absence of established in vivo evidence of tissue regeneration in the adult cerebellum.Here, this BG population was analysed in the adult cerebellum of different species and showed conservation of NSC-associated marker expression including Sox1, Sox2 and Sox9, in chick, primate and mouse cerebellum tissue. NSC-like cells isolated from adult mouse cerebellum showed slower growth when compared to lateral ventricle NSC, as well as differences upon differentiation. In a mouse model of cerebellar degeneration, progressive Purkinje cell loss was linked to cerebellar cortex disorganisation and a significant increase in Sox-positive cells compared to matching controls. These results show that this Sox-positive population responds to cerebellar tissue disruption, suggesting it may represent a mobilisable cellular resource for targeted strategies to promote tissue repair.

Sequential fate-switches in stem-like cells drive the tumorigenic trajectory from human neural stem cells to malignant glioma

Glioblastoma (GBM) is an incurable and highly heterogeneous brain tumor, originating from human neural stem/progenitor cells (hNSCs/hNPCs) years ahead of diagnosis. Despite extensive efforts to characterize hNSCs and end-stage GBM at bulk and single-cell levels, the de novo gliomagenic path from hNSCs is largely unknown due to technical difficulties in early-stage sampling and preclinical modeling. Here, we established two highly penetrant hNSC-derived malignant glioma models, which resemble the histopathology and transcriptional heterogeneity of human GBM. Integrating time-series analyses of whole-exome sequencing, bulk and single-cell RNA-seq, we reconstructed gliomagenic trajectories, and identified a persistent NSC-like population at all stages of tumorigenesis. Through trajectory analyses and lineage tracing, we showed that tumor progression is primarily driven by multi-step transcriptional reprogramming and fate-switches in the NSC-like cells, which sequentially generate malignant heterogeneity and induce tumor phenotype transitions. We further uncovered stage-specific oncogenic cascades, and among the candidate genes we functionally validated C1QL1 as a new glioma-promoting factor. Importantly, the neurogenic-to-gliogenic switch in NSC-like cells marks an early stage characterized by a burst of oncogenic alterations, during which transient AP-1 inhibition is sufficient to inhibit gliomagenesis. Together, our results reveal previously undercharacterized molecular dynamics and fate choices driving de novo gliomagenesis from hNSCs, and provide a blueprint for potential early-stage treatment/diagnosis for GBM.

ETMR-05. SINGLE-CELL RNA-SEQ OF ETMR REVEALS CELL PROGRAMS OF DEVELOPMENTAL HIERARCHY AND CELLULAR DIVERSITY IN THE TUMOR MICROENVIRONMENT

Embryonal tumors with multilayered rosettes (ETMR) are deadly brain malignancies affecting young children. No standard treatment is available and the median survival is less than 12 months. Molecularly, the disease is characterized by the miRNA C19MC cluster amplification, with the expression of multiples miRNAs related to a stem cell program. The discoveries on the purely molecular mechanisms of the disease did not help to create a bridge for new treatment strategies so far and the cellular diversity of ETMR remains poorly understood. In this study, we used single-cell RNA sequencing of murine and human tumors to describe ETMR cellular heterogeneity. Our findings support that intra-tumoral heterogeneity is mainly characterized by 4 cellular programs defining a developmental hierarchy related to different metabolic states: 1) Early quiescent NSC-like cells supported by fatty-acid oxidation 2) Late NSC and NP-like proliferative cells fueled by glycolytic metabolism; 3) Post-mitotic neuroblast-like cells, relying on oxidative-phosphorylation; 4) NSC-like proliferative cells, with metabolic plasticity and capable of performing the three types of metabolism. Tumor-specific ligand-receptor interaction analysis revealed that ETMR exchange with microglia and vascular mural cells (MC) signals related to extracellular matrix (ECM) organization (Cxcl12-CxCr4), stem cell signaling (BMPs-BMP receptors), anti-apoptosis and survival (Ntf3-Ntrk), not seen in the control brain. In addition, the vascular MC showed a cancer-associated fibroblast (CAF) phenotype, with potential prognostic implications, as previously demonstrated for other tumors. This study provides new findings to build up a more robust understanding of ETMR biology and opens space for further studies in the field.

Small molecule-based lineage switch of human adipose-derived stem cells into neural stem cells and functional GABAergic neurons

Cellular reprogramming using small molecules (SMs) without genetic modification provides a promising strategy for generating target cells for cell-based therapy. Human adipose-derived stem cells (hADSCs) are a desirable cell source for clinical application due to their self-renewal capacity, easy obtainability and the lack of safety concerns, such as tumor formation. However, methods to convert hADSCs into neural cells, such as neural stem cells (NSCs), are inefficient, and few if any studies have achieved efficient reprogramming of hADSCs into functional neurons. Here, we developed highly efficient induction protocols to generate NSC-like cells (iNSCs), neuron-like cells (iNs) and GABAergic neuron-like cells (iGNs) from hADSCs via SM-mediated inhibition of SMAD signaling without genetic manipulation. All induced cells adopted morphological, molecular and functional features of their bona fide counterparts. Electrophysiological data demonstrated that iNs and iGNs exhibited electrophysiological properties of neurons and formed neural networks in vitro. Microarray analysis further confirmed that iNSCs and iGNs underwent lineage switch toward a neural fate. Together, these studies provide rapid, reproducible and robust protocols for efficient generation of functional iNSCs, iNs and iGNs from hADSCs, which have utility for modeling disease pathophysiology and providing cell-therapy sources of neurological disorders.

Cells Expressing Prominin-1 in Neonatal Murine Inferior Colliculus Differentiate into Neurons and Glia

Inferior colliculus (IC) is a major center for the integration and processing of acoustic information from ascending auditory pathways. Damage to the IC as well as normal aging can impair auditory function. Novel strategies such as stem cell (SC)-based regenerative therapy are required for functional recovery because mature neural cells have a minimal regenerative capacity after an injury. However, it is not known if there are neural stem cells (NSCs) in the IC. Herein, we screened for NSCs by surface marker analysis using flow cytometry. Isolated IC cells expressing prominin-1 (CD133) exhibited the cardinal NSC properties self-renewal capacity, expression of known NSC markers (SOX2 and nestin), and multipotency. Prominin-1-expressing cells from neonatal IC generated neurospheres, and culture of these neurospheres in differentiation-conditioned medium gave rise to gamma-aminobutyric acid-ergic (GABAergic) neurons, astrocytes, and oligodendrocytes. The presence of NSC-like cells in the IC has important implications for understanding IC development and for potential regenerative therapy.

Safety of neural stem cell transplantation in patients with severe traumatic brain injury.

Neural stem cell (NSC) therapy is a promising treatment for traumatic brain injury (TBI). In addition, mesenchymal stem cells (MSCs) have been investigated for the treatment of TBI due to their functions in neural regeneration and their neurotrophic effect. In the present study, the safety, feasibility and biological effects of autologous MSC-derived NSC-like cell transplantation were investigated in 10 patients with severe TBI. All patients received intravenous or intrathecal injections of human NSC-like cells and were evaluated with physical and neurological examinations, routine laboratory tests and neuroradiological findings. The results indicated that the majority of patients experienced improved neurological function in different degrees during the follow-up period. No mortality or serious adverse events were observed in any patient subsequent to transplantation. Higher serum levels of nerve growth factor and brain-derived neurotrophic factor were detected following the transplantation, as compared with the levels prior to treatment. Overall, the present results suggest that transplantation of autologous NSC-like cells is feasible and appears to be safe for the treatment of non-acute severe TBI.

Conversion of Adipose Tissue-Derived Mesenchymal Stem Cells to Neural Stem Cell-Like Cells by a Single Transcription Factor, Sox2.

Adipose tissue is an attractive source of easily accessible adult candidate cells for regenerative medicine. Adipose tissue-derived mesenchymal stem cells (ADSCs) have multipotency and strong proliferation and differentiation capabilities in vitro. However, as mesodermal multipotent stem cells, whether the ADSCs can convert into induced neural stem cells (NSCs) has so far not been demonstrated. In this study, we found that normally the naïve ADSCs cultured as either monolayer or spheres in NSC medium did not express Sox2 and Pax6 genes and proteins, and could not differentiate to neuron-like cells. However, when we introduced the Sox2 gene into ADSCs by retrovirus, they exhibited a typical NSC-like morphology, and could be passaged continuously, and expressed NSC specific markers Sox2 and Pax6. In addition, the ADSC-derived NSC-like cells displayed the ability to differentiate into neuron-like cells when switched to the differentiation culture medium, expressing neuronal markers, including Tuj1 and MAP2 genes and proteins. Our results suggest the ADSCs can be converted into induced NSC-like cells with a single transcription factor Sox2. This finding could provide another alternative cell source for cell therapy of neurological disorders.

Abstract 1944: Divergent effects of BRAF activation in neural stem and progenitor-like glioblastoma cells

Abstract Glioblastoma multiforme (GBM) are heterogeneous tumors that are refractory to radiation and traditional chemotherapeutics. Separate studies have identified populations of neural stem and progenitor -like GBM cells, however the contribution of these cells types to tumor progression and the response to targeted therapy are poorly understood. Small molecules (such as vemurafenib) targeting the most common MAPK-activating mutation, BRAFV600E, are currently entering clinical trials for treatment of high-grade glioma. Understanding the contribution of different cell types to treatment responses will be necessary for the design of effective adjuvant therapeutic strategies. We show that activating MAPK pathway gene mutations in GBM correlate with elevated expression levels of oligodendrocyte precursor (OPC) and neural stem cell (NSC)-associated genes. Cells expressing CD133 and NG2 coexist in human GBM and BRafV600E expressing mouse neurosphere cultures, and maintain some of their respective neural stem and progenitor-like properties. In isogenic mouse cells expressing BRafV600E, NG2+ cells expand rapidly and symmetrically, while rates of asymmetric division are unaffected in slow-proliferating CD133+ cells. Tracing of proliferation rate with the membrane-associated dye PKH-26 reveals heterogeneity in the proliferation rate of CD133+ cells, but not proliferative NG2+ cells. BRAF-targeted inhibition of human GBM cells with PLX4720, the tool compound of vemurafenib, reduces S-phase entry of NG2+ cells, yet CD133+ cells are less affected. Interestingly targeted inhibition of components of the asymmetric division machinery selectively disrupts G2-M progression in CD133+ cells. The functionally divergent populations of CD133-expressing NSC-like cells will likely be therapeutically problematic with mono-therapeutic approaches. These data point to a differential regulation of key cell cycle processes between CD133+ stem-like cells and NG2+ OPC-like cells, that will inform the intelligent design of compound treatment strategies. Note: This abstract was not presented at the meeting. Citation Format: Robin G. Lerner, Yuichiro Ihara, Kate Lewis, Amelie Griveau, Brian Reichhold, Dian Qu, Martin McMahon, David Rowitch, Charles D. James, Claudia Petritsch. Divergent effects of BRAF activation in neural stem and progenitor-like glioblastoma cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1944. doi:10.1158/1538-7445.AM2014-1944

Efficient Generation of Neural Stem Cell-Like Cells from Rat Adipose Derived Stem Cells After Lentiviral Transduction with Green Fluorescent Protein

Neural stem cells (NSCs) can be isolated from nervous tissues or derived from embryonic stem cells. However, their procurement for clinical applications is limited, and there is a need for alternative types of cell that have NSCs properties. In the present study, the differentiation potential of rat adipose-derived stem cells (ADSCs) was evaluated by infecting these cells with a lentiviral vector-encoding green fluorescent protein (GFP). ADSCs transduced with lentivirus were able to generate NSC-like cells, without any effects on their growth, phenotype, and normal differentiation potential. NSC-like cells derived from ADSCs formed neurospheres and expressed high levels of the neural progenitor marker nestin. In the absence of selected growth factors, these neurospheres differentiated into neurons expressing NeuN and MAP2 and GFAP-expressing glia, as determined by immunocytochemistry, Western blotting, and quantitative real-time polymerase chain reaction. These results demonstrate that ADSCs can be induced to generate neurospheres that have NSC-like properties and may thus constitute a potential source of cells in stem cell therapy for neurological disorders.

Cerebellar neural stem cells differentiate into two distinct types of astrocytes in response to CNTF and BMP2

Neural stem cells (NSCs) are present in postnatal murine cerebellum. The detailed characteristics of these NSCs have never been reported. This study isolated NSC-like cells from postnatal mouse cerebellum. These cells proliferated in response to epidermal growth factor, expressed various NSC markers, and had the ability to self-renew. Neurosphere assays revealed that these cells could differentiate into neurons, astrocytes, and oligodendrocytes, indicating multipotency as NSCs. Although possessing multipotency, most of these cells differentiated into astrocytes spontaneously in vitro. Both ciliary neurotrophic factor (CNTF) and bone morphogenetic protein 2 (BMP2) facilitated expression of glial fibrillary acidic protein (GFAP) and some other characteristics of mature astrocytes by these cells. However, the shape and expression of glutamine transporter GLT-1 of GFAP+ cells generated in the presence of CNTF or BMP2 differed significantly, suggesting that CNTF and BMP2 induced differentiation of these NSCs into two distinct types of astrocytes.

Postnatal age governs the extent of differentiation of hippocampal CA1 and CA3 subfield neural stem/progenitor cells into neurons and oligodendrocytes

While neural stem/progenitor cells (NSCs) in the dentate gyrus of the hippocampus have been extensively characterized, the behavior of NSCs in the CA1 and CA3 subfields of the hippocampus is mostly unclear. Therefore, we compared the in vitro behavior of NSCs expanded from the micro-dissected CA1 and CA3 subfields of postnatal day (PND) 4 and 12 Fischer 344 rats. A small fraction (∼1%) of dissociated cells from CA1 and CA3 subfields of both PND 4 and 12 hippocampi formed neurospheres in the presence of EGF and FGF-2. A vast majority of neurosphere cells expressed NSC markers such as nestin, Sox-2 and Musashi-1. Differentiation assays revealed the ability of these NSCs to give rise to neurons, astrocytes, and oligodendrocytes. Interestingly, the overall neuronal differentiation of NSCs from both subfields decreased with age (23–28% at PND4 to 5–10% at PND12) but the extent of oligodendrocyte differentiation from NSCs increased with age (24–32% at PND 4 to 45–55% at PND 12). Differentiation of NSCs into astrocytes was however unchanged (40–48%). Furthermore, NSCs from both subfields gave rise to GABA-ergic neurons including subclasses expressing markers such as calbindin, calretinin, neuropeptide Y and parvalbumin. However, the fraction of neurons that expressed GABA decreased between PND4 (59–67%) and PND 12 (25–38%). Additional analyses revealed the presence of proliferating NSC-like cells (i.e. cells expressing Ki-67 and Sox-2) in different strata of hippocampal CA1 and CA3 subfields of both PND4 and PND 12 animals. Thus, multipotent NSCs persist in both CA1 and CA3 subfields of the hippocampus in the postnatal period. Such NSCs also retain their ability to give rise to both GABA-ergic and non-GABA-ergic neurons. However, their overall neurogenic potential declines considerably in the early postnatal period.

Neural stem cell-like cells derived from autologous bone mesenchymal stem cells for the treatment of patients with cerebral palsy

BackgroundStem cell therapy is a promising treatment for cerebral palsy, which refers to a category of brain diseases that are associated with chronic motor disability in children. Autologous MSCs may be a better cell source and have been studied for the treatment of cerebral palsy because of their functions in tissue repair and the regulation of immunological processes.MethodsTo assess neural stem cell–like (NSC-like) cells derived from autologous marrow mesenchymal stem cells as a novel treatment for patients with moderate-to-severe cerebral palsy, a total of 60 cerebral palsy patients were enrolled in this open-label, non-randomised, observer-blinded controlled clinical study with a 6-months follow-up. For the transplantation group, a total of 30 cerebral palsy patients received an autologous NSC-like cells transplantation (1-2 × 107 cells into the subarachnoid cavity) and rehabilitation treatments whereas 30 patients in the control group only received rehabilitation treatment.ResultsWe recorded the gross motor function measurement scores, language quotients, and adverse events up to 6 months post-treatment. The gross motor function measurement scores in the transplantation group were significantly higher at month 3 (the score increase was 42.6, 95% CI: 9.8–75.3, P=.011) and month 6 (the score increase was 58.6, 95% CI: 25.8–91.4, P=.001) post-treatment compared with the baseline scores. The increase in the Gross Motor Function Measurement scores in the control group was not significant. The increases in the language quotients at months 1, 3, and 6 post-treatment were not statistically significant when compared with the baseline quotients in both groups. All the 60 patients survived, and none of the patients experienced serious adverse events or complications.ConclusionOur results indicated that NSC-like cells are safe and effective for the treatment of motor deficits related to cerebral palsy. Further randomised clinical trials are necessary to establish the efficacy of this procedure.

Activation of neural progenitor cells in human eyes with proliferative vitreoretinopathy

In addition to the ability for self-renewal and functional differentiation, neural stem/progenitor cells (NSCs) can respond to CNS injuries by targeted migration. In lower vertebrates, retinal injury is known to activate NSCs in the ciliary marginal zone (CMZ). Cells expressing markers of NSCs are also present in the ciliary body epithelium (CE) and in Müller glia in the peripheral retina (PR) of the adult human eye. However, these cells seem to be quiescent in the adult human eye and recent reports have shown that CE cells have limited properties of NSCs. In order to further clarify whether NSCs exist in the adult human eye, we tested whether NSC-like cells could be activated in eyes with proliferative vitreoretinopathy (PVR). The PR and CE were studied for NSC-associated markers in human enucleated control eyes and eyes with confirmed PVR, as well as in a mouse model of PVR. Furthermore, cells isolated from vitreous samples obtained during vitrectomies for retinal detachment were directly fixed or cultured in a stem cell-promoting medium and compared to cells cultured from the post-mortem retina and CE. In situ characterization of the normal eyes revealed robust expression of markers present in NSCs (Nestin, Sox2, Pax6) only around peripheral cysts of the proximal pars plana region and the PR, the latter population also staining for the glial marker GFAP. Although there were higher numbers of dividing cells in the CE of PVR eyes than in controls, we did not detect NSC-associated markers in the CE except around the proximal pars plana cysts. In the mice PVR eyes, Nestin activation was also found in the CE. In human PVR eyes, proliferation of both non-glial and glial cells co-staining NSC-associated markers was evident around the ora serrata region. Spheres formed in 7/10 vitreous samples from patients with PVR compared to 2/15 samples from patients with no known PVR, and expressed glial – and NSC-associated markers both after direct fixation and repetitive passages. In conclusion, the adult human eye may harbor two different populations of neuroepithelial stem/progenitor cells; a non-glial population located in the proximal pars plana around peripheral cysts in addition to a population with Müller glia characteristics. Yet, we only found that the glial population was able to respond to retinal injury by targeted migration into the vitreous.

Generation of neural stem cell-like cells from bone marrow-derived human mesenchymal stem cells

Under appropriate culture conditions, bone marrow (BM)-derived mesenchymal stem cells are capable of differentiating into diverse cell types unrelated to their phenotypical embryonic origin, including neural cells. Here, we report the successful generation of neural stem cell (NSC)-like cells from BM-derived human mesenchymal stem cells (hMSCs). Initially, hMSCs were cultivated in a conditioned medium of human neural stem cells. In this culture system, hMSCs were induced to become NSC-like cells, which proliferate in neurosphere-like structures and express early NSC markers. Like central nervous system-derived NSCs, these BM-derived NSC-like cells were able to differentiate into cells expressing neural markers for neurons, astrocytes, and oligodendrocytes. Whole-cell patch clamp recording revealed that neuron-like cells, differentiated from NSC-like cells, exhibited electrophysiological properties of neurons, including action potentials. Transplantation of NSC-like cells into mouse brain confirmed that these NSC-like cells retained their capability to differentiate into neuronal and glial cells in vivo. Our data show that multipotent NSC-like cells can be efficiently produced from BM-derived hMSCs in culture and that these cells may serve as a useful alternative to human neural stem cells for potential clinical applications such as autologous neuroreplacement therapies.

Bmi1 puSHHes reprogramming

In 2006, the group of Shinya Yamanaka demonstrated that somatic cells could be reprogrammed into induced pluripotent stem cells (iPSCs) by ectopic expression of four transcription factors associated to stemness: Oct4, Sox2, Klf4 and c-Myc 1. This groundbreaking discovery opened the possibility of generating patient-specific cells for research, drug development and regenerative medicine. Due to the tremendous potential of its clinical applications, understanding the process of reprogramming has become a priority and one of the most fascinating biomedical research topics. In the last five years, scientists around the world are racing to solve two main issues regarding reprogramming. The first one is technical and consists in improving the efficiency of reprogramming and avoiding the necessity to integrate exogenous DNA into the reprogrammed host cells. Several advances have been made over the last years, both in replacing individual factors with chemicals, and in eliminating the need to alter the genome of the host cell 2. However, pure chemical reprogramming has not yet been achieved despite intense efforts, and integration-free reprogramming is still poorly efficient 2. Another major focus of research is to understand the molecular mechanisms underlying reprogramming. However, the low efficiency of the process makes this task challenging. Recently, our group reported that the Ink4a/Arf locus is a major barrier for reprogramming 3. For example, complementation of the Yamanaka factors with an shRNA against the Ink4a/Arf locus greatly improves reprogramming efficiency 3. Bmi1 is part of the Polycomb Repressive Complex 1 (PRC1) involved in transcriptional silencing through heterochromatinization. The Bmi1 gene is a well-characterized oncogene that silences the Ink4a/Arf locus and cooperates with c-Myc in lymphomagenesis 4 and it is overexpressed in several human cancers 5. In addition to this, Bmi1 has been shown over the years to be a key regulator of self-renewal in multiple adult stem cells including hematopoietic stem cells (HSCs), neural stem cells (NSCs) and, more recently, prostate stem cells (PrSCs) 5, 6. Sonic hedgehog (Shh) signaling is a major developmental signaling pathway and Bmi1 together with N-Myc and cyclin D2 are major downstream effectors of Shh for proliferation and self-renewal of cerebellar progenitor cells 5. Based on the impact of Bmi1 on the Ink4a/Arf locus and on stem cells, one might speculate what is the role of Bmi1 in reprogramming? In a recent paper published in Cell Research, Moon et al. 7 explore the role of Bmi1 in reprogramming. When overexpressing Bmi1 in mouse embryonic fibroblasts (MEFs), the authors show a marked increase in the levels of endogenous Sox2, N-Myc and Klf4, together with drastically reduced levels of Ink4a, Arf and p53. In line with this, Moon et al. demonstrate that iPSCs can be generated from MEFs using a simplified reprogramming cocktail consisting only of Bmi1 and Oct4, and with comparable efficiency to the Yamanaka three-factor cocktail (Sox2, Klf4 and Oct4). This is an important and satisfying finding, however, the oncogenic activity of Bmi1 is not very appealing for future therapeutic applications. Therefore, Moon et al. took their findings one step further. As mentioned above, the Shh signaling pathway was known to induce Bmi1 expression in neural progenitors 5. They treated MEFs with Shh and exposed them to NSC culture medium. Using this protocol, MEFs were transdifferentiated into NSC-like cells, a process that was also recapitulated by overexpression of Bmi1 7. Neural stem cells were previously known for being reprogrammed only with Oct4 due to their high endogenous levels of Sox2 and Klf4 8. Therefore, Moon et al. transduced Oct4 into the Shh-induced NSC-like cells and were able to obtain iPSCs. This takes the authors to their last tour de force by which they reprogram MEFs with the combination of two chemical Shh agonists (oxysterol and purmorphamine) and Oct4 7. The above findings illuminate the process of reprogramming and also get us closer to chemical reprogramming. The advantage of a hypothetical chemical reprogramming resides in its simplicity and safety, which are of high importance for future therapeutic applications. Previously, two other teams have published in Cell Research that combining Oct4 with either BMPs or with a combination of small molecules are sufficient to reprogram somatic cells to iPSCs 9, 10. The new findings by Moon et al. 7 broaden the spectrum of molecules that could be used in combination with Oct4. So far, Oct4 is the only factor that has not been replaced by small molecules. However, based on the speed of progress in this field, it is not unrealistic to anticipate that pure chemical reprogramming will be achieved in the near future.

Direct Generation of Neurosphere-Like Cells from Human Dermal Fibroblasts

Neural stem cell (NSC) transplantation replaces damaged brain cells and provides disease-modifying effects in many neurological disorders. However, there has been no efficient way to obtain autologous NSCs in patients. Given that ectopic factors can reprogram somatic cells to be pluripotent, we attempted to generate human NSC-like cells by reprograming human fibroblasts. Fibroblasts were transfected with NSC line-derived cellular extracts and grown in neurosphere culture conditions. The cells were then analyzed for NSC characteristics, including neurosphere formation, gene expression patterns, and ability to differentiate. The obtained induced neurosphere-like cells (iNS), which formed daughter neurospheres after serial passaging, expressed neural stem cell markers, and had demethylated SOX2 regulatory regions, all characteristics of human NSCs. The iNS had gene expression patterns that were a combination of the patterns of NSCs and fibroblasts, but they could be differentiated to express neuroglial markers and neuronal sodium channels. These results show for the first time that iNS can be directly generated from human fibroblasts. Further studies on their application in neurological diseases are warranted.

Reprogramming fibroblasts into induced pluripotent stem cells with Bmi1

Somatic cells can be reprogrammed into induced pluripotent stem (iPS) cells by the transcription factors Oct4, Sox2, and Klf4 in combination with c-Myc. Recently, Sox2 plus Oct4 was shown to reprogram fibroblasts and Oct4 alone was able to reprogram mouse and human neural stem cells (NSCs) into iPS cells. Here, we report that Bmi1 leads to the transdifferentiation of mouse fibroblasts into NSC-like cells, and, in combination with Oct4, can replace Sox2, Klf4 and c-Myc during the reprogramming of fibroblasts into iPS cells. Furthermore, activation of sonic hedgehog signaling (by Shh, purmorphamine, or oxysterol) compensates for the effects of Bmi1, and, in combination with Oct4, reprograms mouse embryonic and adult fibroblasts into iPS cells. One- and two-factor iPS cells are similar to mouse embryonic stem cells in their global gene expression profile, epigenetic status, and in vitro and in vivo differentiation into all three germ layers, as well as teratoma formation and germline transmission in vivo. These data support that converting fibroblasts with Bmi1 or activation of the sonic hedgehog pathway to an intermediate cell type that expresses Sox2, Klf4, and N-Myc allows iPS generation via the addition of Oct4.

Membrane Properties of Neuron-Like Cells Generated from Adult Human Bone-Marrow-Derived Mesenchymal Stem Cells

Adult mesenchymal stem cells (MeSCs) isolated from human bone marrow are capable of generating neural stem cell (NSC)-like cells that can be subsequently differentiated into cells expressing molecular markers for neurons. Here we report that these neuron-like cells had functional properties similar to those of brain-derived neurons. Whole-cell patch-clamp recordings and calcium imaging experiments were performed on neuron-like cells differentiated from bone-marrow-derived NSC-like cells. The neuron-like cells were subjected to current pulses to determine if they were capable of generating depolarization-induced action potentials. We found that nearly all of the cells with neuron-like morphology exhibited active membrane properties in response to the depolarizing pulses. The most common response was a single spike-like event with an overshoot and brief afterhyperpolarization. Cells that did not generate overshooting spike-like events usually displayed rectifying current-voltage relationships. The prevalence of these active membrane properties in response to the depolarizing current pulses suggested that the human MeSCs (hMeSCs) were capable of converting to a neural lineage under defined culture conditions. The spike-like events were blocked by the voltage-gated sodium channel inhibitor lidocaine, but unaffected by another sodium channel inhibitor tetrodotoxin (TTX). In calcium imaging experiments, the neuron-like cells responded to potassium chloride depolarization and l-glutamate application with increases in the cytoplasmic calcium levels. Thus, the neuron-like cells appeared to express TTX-resistant voltage-gated sodium channels, voltage-gated calcium channels, and functional l-glutamate receptors. These results demonstrate that hMeSCs were capable of generating cells with characteristics typical of functional neurons that may prove useful for neuroreplacement therapies.

Genome-wide expression profiling and functional network analysis upon neuroectodermal conversion of human mesenchymal stem cells suggest HIF-1 and miR-124a as important regulators

Tissue-specific stem cells, such as bone-marrow-derived human mesenchymal stem cells (hMSCs), are thought to be lineage restricted and therefore, could only be differentiated into cell types of the tissue of origin. Several recent studies however have suggested that these types of stem cells might be able to break barriers of germ layer commitment and differentiate in vitro into cells with neuroectodermal properties. We reported earlier about efficient conversion of adult hMSCs into a neural stem cell (NSC)-like population (hmNSCs, for human marrow-derived NSC-like cells) with all major properties of NSCs including functional neuronal differentiation capacity. Here we compared the transcriptomes from hMSCs and hmNSCs using a novel strategy by combining classic Affymetrix oligonucleotide microarray profiling with regulatory and protein interaction network analyses to shed light on regulatory protein networks involved in this neuroectodermal conversion process. We found differential regulation of extracellular matrix protein transcripts, up-regulation of distinct neuroectodermal and NSCs marker genes and local chromosomal transcriptional up-regulation at chromosome 4q13.3. In comparison to hMSCs and primary adult hippocampal NSCs, the transcriptome of hmNSCs displayed minor overlap with both other cell populations. Advanced bioinformatics of regulated genes upon neuroectodermal conversion identified transcription factor networks with HIF-1 and microRNA miR-124a as potential major regulators. Together, transgerminal neuroectodermal conversion of hMSCs into NSC-like cells is accompanied by extensive changes of their global gene expression profile, which might be controlled in part by transcription factor networks related to HIF-1 and miR-124a.