Oct4-induced pluripotency in adult neural stem cells.

The four transcription factors Oct4, Sox2, Klf4, and c-Myc can induce pluripotency in mouse and human fibroblasts. We previously described direct reprogramming of adult mouse neural stem cells (NSCs) by Oct4 and either Klf4 or c-Myc. NSCs endogenously express Sox2, c-Myc, and Klf4 as well as several intermediate reprogramming markers. Here we report that exogenous expression of the germline-specific transcription factor Oct4 is sufficient to generate pluripotent stem cells from adult mouse NSCs. These one-factor induced pluripotent stem cells (1F iPS) are similar to embryonic stem cells in vitro and in vivo. Not only can these cells can be efficiently differentiated into NSCs, cardiomyocytes, and germ cells in vitro, but they are also capable of teratoma formation and germline transmission in vivo. Our results demonstrate that Oct4 is required and sufficient to directly reprogram NSCs to pluripotency.

Etv2- and Fli1-Induced Vascular Progenitor Cells Enhance Functional Recovery in Ischemic Vascular Disease Model-Brief Report.

OBJECTIVE: Vascular progenitor cells (VPCs), which are able to differentiate into both endothelial cells and smooth muscle cells, have the potential for treatment of ischemic diseases. Generated by pluripotent stem cells, VPCs carry the risk of tumorigenicity in clinical application. This issue could be resolved by direct lineage conversion, the induction of functional cells from another lineage by using only lineage-restricted transcription factors. Here, we show that induced VPCs (iVPCs) can be generated from fibroblasts by ETS (E-twenty six) transcription factors, Etv2 and Fli1. Approach and Results: Mouse fibroblasts were infected with lentivirus encoding Etv2 and Fli1. Cell colonies appeared in Fli1- and Etv2/Fli1-infected groups and were mechanically picked. The identity of cell colonies was confirmed by proliferation assay and reverse-transcription polymerase chain reaction with vascular markers. Etv2/Fli1- infected cell colonies were sorted by CD144 (also known as CDH5, VE-cadherin). We defined that CD144-positive iVPCs maintained its own population and expanded stably at multiple passages. iVPCs could differentiate into functional endothelial cells and smooth muscle cells by a defined medium. The functionalities of iVPC-derived endothelial cells and smooth muscle cells were confirmed by analyzing LDL (low-density lipoprotein) uptake, carbachol-induced contraction, and tube formation in vitro. Transplantation of iVPCs into the ischemic hindlimb model enhanced blood flow without tumor formation in vivo. Human iVPCs were generated by human ETS transcription factors ETV2 and FLI1. CONCLUSIONS: We demonstrate that ischemic disease curable iVPCs, which have self-renewal and bipotency, can be generated from mouse fibroblasts by enforced ETS family transcription factors, Etv2 and Fli1 expression. Our simple strategy opens insights into stem cell-based ischemic disease therapy.

Direct monitoring of live human pluripotent stem cells by a highly selective pluripotency sensor.

Human pluripotent stem cells (hPSCs) are a useful cell source for regenerative medicine. Despite having a potential of hPSCs for cell-based therapy, there is a need for a selective human pluripotency sensor for monitoring of live hPSCs. Here, we report the discovery of a novel pluripotency sensor (SHI5) from BODIPY-based library by high-throughput cell-based screening and describe the use of SHI5 to identify and isolate human embryonic stem cells and human induced pluripotent stem cells. We demonstrate that SHI5-based assay can be applied to live cells that gain pluripotency in the reprogramming process without any effect on their viability. We also show that SHI5 is internalized through a clathrin-mediated endocytosis pathway. These findings suggest that SHI5 can be an attractive sensor for pluripotency cells during reprogramming. Taken together, SHI5-based screening for hPSCs opens probably unlimited possibilities of detection probe for hPSC therapy via assures their safety issue.

Oct4-induced oligodendrocyte progenitor cells enhance functional recovery in spinal cord injury model.

The generation of patient-specific oligodendrocyte progenitor cells (OPCs) holds great potential as an expandable cell source for cell replacement therapy as well as drug screening in spinal cord injury or demyelinating diseases. Here, we demonstrate that induced OPCs (iOPCs) can be directly derived from adult mouse fibroblasts by Oct4-mediated direct reprogramming, using anchorage-independent growth to ensure high purity. Homogeneous iOPCs exhibit typical small-bipolar morphology, maintain their self-renewal capacity and OPC marker expression for more than 31 passages, share high similarity in the global gene expression profile to wild-type OPCs, and give rise to mature oligodendrocytes and astrocytes in vitro and in vivo. Notably, transplanted iOPCs contribute to functional recovery in a spinal cord injury (SCI) model without tumor formation. This study provides a simple strategy to generate functional self-renewing iOPCs and yields insights for the in-depth study of demyelination and regenerative medicine.

Erythroid differentiation of human induced pluripotent stem cells is independent of donor cell type of origin.

Epigenetic memory in induced pluripotent stem cells, which is related to the somatic cell type of origin of the stem cells, might lead to variations in the differentiation capacities of the pluripotent stem cells. In this context, induced pluripotent stem cells from human CD34(+) hematopoietic stem cells might be more suitable for hematopoietic differentiation than the commonly used fibroblast-derived induced pluripotent stem cells. To investigate the influence of an epigenetic memory on the ex vivo expansion of induced pluripotent stem cells into erythroid cells, we compared induced pluripotent stem cells from human neural stem cells and human cord blood-derived CD34(+) hematopoietic stem cells and evaluated their potential for differentiation into hematopoietic progenitor and mature red blood cells. Although genome-wide DNA methylation profiling at all promoter regions demonstrates that the epigenetic memory of induced pluripotent stem cells is influenced by the somatic cell type of origin of the stem cells, we found a similar hematopoietic induction potential and erythroid differentiation pattern of induced pluripotent stem cells of different somatic cell origin. All human induced pluripotent stem cell lines showed terminal maturation into normoblasts and enucleated reticulocytes, producing predominantly fetal hemoglobin. Differences were only observed in the growth rate of erythroid cells, which was slightly higher in the induced pluripotent stem cells derived from CD34(+) hematopoietic stem cells. More detailed methylation analysis of the hematopoietic and erythroid promoters identified similar CpG methylation levels in the induced pluripotent stem cell lines derived from CD34(+) cells and those derived from neural stem cells, which confirms their comparable erythroid differentiation potential.

Direct reprogramming of human neural stem cells by OCT4.

Induced pluripotent stem (iPS) cells have been generated from mouse and human somatic cells by ectopic expression of four transcription factors (OCT4 (also called POU5F1), SOX2, c-Myc and KLF4). We previously reported that Oct4 alone is sufficient to reprogram directly adult mouse neural stem cells to iPS cells. Here we report the generation of one-factor human iPS cells from human fetal neural stem cells (one-factor (1F) human NiPS cells) by ectopic expression of OCT4 alone. One-factor human NiPS cells resemble human embryonic stem cells in global gene expression profiles, epigenetic status, as well as pluripotency in vitro and in vivo. These findings demonstrate that the transcription factor OCT4 is sufficient to reprogram human neural stem cells to pluripotency. One-factor iPS cell generation will advance the field further towards understanding reprogramming and generating patient-specific pluripotent stem cells.

Pluripotent stem cells induced from adult neural stem cells by reprogramming with two factors.

Reprogramming of somatic cells is a valuable tool to understand the mechanisms of regaining pluripotency and further opens up the possibility of generating patient-specific pluripotent stem cells. Reprogramming of mouse and human somatic cells into pluripotent stem cells, designated as induced pluripotent stem (iPS) cells, has been possible with the expression of the transcription factor quartet Oct4 (also known as Pou5f1), Sox2, c-Myc and Klf4 (refs 1-11). Considering that ectopic expression of c-Myc causes tumorigenicity in offspring and that retroviruses themselves can cause insertional mutagenesis, the generation of iPS cells with a minimal number of factors may hasten the clinical application of this approach. Here we show that adult mouse neural stem cells express higher endogenous levels of Sox2 and c-Myc than embryonic stem cells, and that exogenous Oct4 together with either Klf4 or c-Myc is sufficient to generate iPS cells from neural stem cells. These two-factor iPS cells are similar to embryonic stem cells at the molecular level, contribute to development of the germ line, and form chimaeras. We propose that, in inducing pluripotency, the number of reprogramming factors can be reduced when using somatic cells that endogenously express appropriate levels of complementing factors.

Differentiation efficiency of induced pluripotent stem cells depends on the number of reprogramming factors.

Reprogramming of somatic cells into induced pluripotent stem cells (iPSCs) by retroviral overexpression of the transcription factors Oct4, Sox2, Klf4, and c-Myc holds great promise for the development of personalized cell replacement therapies. In an attempt to minimize the risk for chromosomal disruption and to simplify reprogramming, several studies demonstrated that a reduced set of reprogramming factors is sufficient to generate iPSC, albeit at lower efficiency. To elucidate the influence of factor reduction on subsequent differentiation, we compared the efficiency of neuronal differentiation in iPSC generated from postnatal murine neural stem cells with either one (Oct4; iPSC(1F-NSC) ), two (Oct4, Klf4; iPSC(2F-NSC) ), or all four factors (iPSC(4F-NSC) ) with those of embryonic stem cells (ESCs) and iPSC produced from fibroblasts with all four factors (iPSC(4F-MEF) ). After 2 weeks of coculture with PA6 stromal cells, neuronal differentiation of iPSC(1F-NSC) and iPSC(2F-NSC) was less efficient compared with iPSC(4F-NSC) and ESC, yielding lower proportions of colonies that stained positive for early and late neuronal markers. Electrophysiological analyses after 4 weeks of differentiation identified functional maturity in neurons differentiated from ESC, iPSC(2F-NSC) , iPSC(4F-NSC) , and iPSC(4F-MEF) but not in those from iPSC(1F-NSC) . Similar results were obtained after hematoendothelial differentiation on OP9 bone marrow stromal cells, where factor-reduced iPSC generated lower proportions of colonies with hematoendothelial progenitors than colonies of ESC, iPSC(4F-NSC) , and iPSC(4F-MEF) . We conclude that a reduction of reprogramming factors does not only reduce reprogramming efficiency but may also worsen subsequent differentiation and hinder future application of iPSC in cell replacement therapies.

Human skeletal muscle organoids model fetal myogenesis and sustain uncommitted PAX7 myogenic progenitors.

In vitro culture systems that structurally model human myogenesis and promote PAX7+ myogenic progenitor maturation have not been established. Here we report that human skeletal muscle organoids can be differentiated from induced pluripotent stem cell lines to contain paraxial mesoderm and neuromesodermal progenitors and develop into organized structures reassembling neural plate border and dermomyotome. Culture conditions instigate neural lineage arrest and promote fetal hypaxial myogenesis toward limb axial anatomical identity, with generation of sustainable uncommitted PAX7 myogenic progenitors and fibroadipogenic (PDGFRa+) progenitor populations equivalent to those from the second trimester of human gestation. Single-cell comparison to human fetal and adult myogenic progenitor /satellite cells reveals distinct molecular signatures for non-dividing myogenic progenitors in activated (CD44High/CD98+/MYOD1+) and dormant (PAX7High/FBN1High/SPRY1High) states. Our approach provides a robust 3D in vitro developmental system for investigating muscle tissue morphogenesis and homeostasis.

Humans contains around 650 skeletal muscles which allow the body to move around and maintain its posture. Skeletal muscles are made up of individual cells that bundle together into highly organized structures. If this group of muscles fail to develop correctly in the embryo and/or fetus, this can lead to muscular disorders that can make it painful and difficult to move. One way to better understand how skeletal muscles are formed, and how this process can go wrong, is to grow them in the laboratory. This can be achieved using induced pluripotent stem cells (iPSCs), human adult cells that have been 'reprogrammed' to behave like cells in the embryo that can develop in to almost any cell in the body. The iPSCs can then be converted into specific cell types in the laboratory, including the cells that make up skeletal muscle. Here, Mavrommatis et al. created a protocol for developing iPSCs into three-dimensional organoids which resemble how cells of the skeletal muscle look and arrange themselves in the fetus. To form the skeletal muscle organoid, Mavrommatis et al. treated iPSCs that were growing in a three-dimensional environment with various factors that are found early on in development. This caused the iPSCs to organize themselves in to embryonic and fetal structures that will eventually give rise to the parts of the body that contain skeletal muscle, such as the limbs. Within the organoid were cells that produced Pax7, a protein commonly found in myogenic progenitors that specifically mature into skeletal muscle cells in the fetus. Pax 7 is also present in 'satellite cells' that help to regrow damaged skeletal muscle in adults. Indeed, Mavrommatis et al. found that the myogenic progenitors produced by the organoid were able to regenerate muscle when transplanted in to adult mice. These findings suggest that this organoid protocol can generate cells that will give rise to skeletal muscle. In the future, these lab-grown progenitors could potentially be created from cells isolated from patients and used to repair muscle injuries. The organoid model could also provide new insights in to how skeletal muscles develop in the fetus, and how genetic mutations linked with muscular disorders disrupt this process.

Induced Endothelial Cell-Integrated Liver Assembloids Promote Hepatic Maturation and Therapeutic Effect on Cholestatic Liver Fibrosis.

The transplantation of pluripotent stem cell (PSC)-derived liver organoids has been studied to solve the current donor shortage. However, the differentiation of unintended cell populations, difficulty in generating multi-lineage organoids, and tumorigenicity of PSC-derived organoids are challenges. However, direct conversion technology has allowed for the generation lineage-restricted induced stem cells from somatic cells bypassing the pluripotent state, thereby eliminating tumorigenic risks. Here, liver assembloids (iHEAs) were generated by integrating induced endothelial cells (iECs) into the liver organoids (iHLOs) generated with induced hepatic stem cells (iHepSCs). Liver assembloids showed enhanced functional maturity compared to iHLOs in vitro and improved therapeutic effects on cholestatic liver fibrosis animals in vivo. Mechanistically, FN1 expressed from iECs led to the upregulation of Itgα5/ß1 and Hnf4α in iHEAs and were correlated to the decreased expression of genes related to hepatic stellate cell activation such as Lox and Spp1 in the cholestatic liver fibrosis animals. In conclusion, our study demonstrates the possibility of generating transplantable iHEAs with directly converted cells, and our results evidence that integrating iECs allows iHEAs to have enhanced hepatic maturation compared to iHLOs.

Conversion of mouse epiblast stem cells to an earlier pluripotency state by small molecules.

Epiblast stem cells (EpiSCs) are pluripotent cells derived from post-implantation late epiblasts in vitro. EpiSCs are incapable of contributing to chimerism, indicating that EpiSCs are less pluripotent and represent a later developmental pluripotency state compared with inner cell mass stage murine embryonic stem cells (mESCs). Using a chemical approach, we found that blockage of the TGFß pathway or inhibition of histone demethylase LSD1 with small molecule inhibitors induced dramatic morphological changes in EpiSCs toward mESC phenotypes with simultaneous activation of inner cell mass-specific gene expression. However, full conversion of EpiSCs to the mESC-like state with chimerism competence could be readily generated only with the combination of LSD1, ALK5, MEK, FGFR, and GSK3 inhibitors. Our results demonstrate that appropriate synergy of epigenetic and signaling modulations could convert cells at the later developmental pluripotency state to the earlier mESC-like pluripotency state, providing new insights into pluripotency regulation.

Effects of neural progenitor cells on sensorimotor recovery and endogenous repair mechanisms after photothrombotic stroke.

BACKGROUND AND PURPOSE: Intravenous neural progenitor cell (NPC) treatment was shown to improve functional recovery after experimental stroke. The underlying mechanisms, however, are not completely understood so far. Here, we investigated the effects of systemic NPC transplantation on endogenous neurogenesis and dendritic plasticity of host neurons. METHODS: Twenty-four hours after photothrombotic ischemia, adult rats received either 5 million NPC or placebo intravenously. Behavioral tests were performed weekly up to 4 weeks after ischemia. Endogenous neurogenesis, dendritic length, and dendritic branching of cortical pyramid cells and microglial activation were quantified. RESULTS: NPC treatment led to a significantly improved sensorimotor function measured by the adhesive removal test. The dendritic length and the amount of branch points were significantly increased after NPC transplantation, whereas endogenous neurogenesis was decreased compared to placebo therapy. Decreased endogenous neurogenesis was associated with an increased number of activated microglial cells. CONCLUSIONS: Our findings suggest that an increased dendritic plasticity might be the structural basis of NPC-induced functional recovery. The decreased endogenous neurogenesis after NPC treatment seems to be mediated by microglial activation.

Synergistic control of mechanics and microarchitecture of 3D bioactive hydrogel platform to promote the regenerative potential of engineered hepatic tissue.

Culturing autologous cells with therapeutic potential derived from a patient within a bioactive scaffold to induce functioning tissue formation is considered the ideal methodology towards realizing patient-specific regenerative medicine. Hydrogels are often employed as the scaffold material for this purpose mainly for their tunable mechanical and diffusional properties as well as presenting cell-responsive moieties. Herein, a two-fold strategy was employed to control the physicomechanical properties and microarchitecture of hydrogels to maximize the efficacy of engineered hepatic tissues. First, a hydrophilic polymeric crosslinker with a tunable degree of reactive functional groups was employed to control the mechanical properties in a wide range while minimizing the change in diffusional properties. Second, photolithography technique was utilized to introduce microchannels into hydrogels to overcome the critical diffusional limit of bulk hydrogels. Encapsulating hepatic progenitor cells derived via direct reprogramming of tissue-harvested fibroblasts, the application of this strategy to control the mechanics, diffusion, and architecture of hydrogels in a combinatorial manner could allow the optimization of their hepatic functions. The regenerative capacity of this engineered hepatic tissue was further demonstrated using an in vivo acute liver injury model.

Stable isotope labeling by amino acids in cell culture (SILAC) and proteome quantitation of mouse embryonic stem cells to a depth of 5,111 proteins.

Embryonic stem (ES) cells are pluripotent cells isolated from mammalian preimplantation embryos. They are capable of differentiating into all cell types and therefore hold great promise in regenerative medicine. Here we show that murine ES cells can be fully SILAC (stable isotope labeling by amino acids in cell culture)-labeled when grown feeder-free during the last phase of cell culture. We fractionated the SILAC-labeled ES cell proteome by one-dimensional gel electrophoresis and by isoelectric focusing of peptides. High resolution analysis on a linear ion trap-orbitrap instrument (LTQ-Orbitrap) at sub-ppm mass accuracy resulted in confident identification and quantitation of more than 5,000 distinct proteins. This is the largest quantified proteome reported to date and contains prominent stem cell markers such as OCT4, NANOG, SOX2, and UTF1 along with the embryonic form of RAS (ERAS). We also quantified the proportion of the ES cell proteome present in cytosolic, nucleoplasmic, and membrane/chromatin fractions. We compared two different preparation approaches, cell fractionation followed by one-dimensional gel separation and in-solution digestion of total cell lysate combined with isoelectric focusing, and found comparable proteome coverage with no apparent bias for any functional protein classes for either approach. Bioinformatics analysis of the ES cell proteome revealed a broad distribution of cellular functions with overrepresentation of proteins involved in proliferation. We compared the proteome with a recently published map of chromatin states of promoters in ES cells and found excellent correlation between protein expression and the presence of active and repressive chromatin marks.

SPON1 Can Reduce Amyloid Beta and Reverse Cognitive Impairment and Memory Dysfunction in Alzheimer's Disease Mouse Model.

Alzheimer's disease (AD) is a complex, age-related neurodegenerative disease that is the most common form of dementia. However, the cure for AD has not yet been founded. The accumulation of amyloid beta (Aß) is considered to be a hallmark of AD. Beta-site amyloid precursor protein cleaving enzyme 1 (BACE1), also known as beta secretase is the initiating enzyme in the amyloidogenic pathway. Blocking BACE1 could reduce the amount of Aß, but this would also prohibit the other functions of BACE1 in brain physiological activity. SPONDIN1 (SPON1) is known to bind to the BACE1 binding site of the amyloid precursor protein (APP) and blocks the initiating amyloidogenesis. Here, we show the effect of SPON1 in Aß reduction in vitro in neural cells and in an in vivo AD mouse model. We engineered mouse induced neural stem cells (iNSCs) to express Spon1. iNSCs harboring mouse Spon1 secreted SPON1 protein and reduced the quantity of Aß when co-cultured with Aß-secreting Neuro 2a cells. The human SPON1 gene itself also reduced Aß in HEK 293T cells expressing the human APP transgene with AD-linked mutations through lentiviral-mediated delivery. We also demonstrated that injecting SPON1 reduced the amount of Aß and ameliorated cognitive dysfunction and memory impairment in 5xFAD mice expressing human APP and PSEN1 transgenes with five AD-linked mutations.

Decellularized extracellular matrix-based bio-ink with enhanced 3D printability and mechanical properties.

Recently, decellularized extracellular matrix-based bio-ink (dECM bio-ink) derived from animal organs is attracting attention because of its excellent biocompatibility. However, its poor 3D printability and weak mechanical properties remain a challenge. Here, we developed a new dECM bio-ink with enhanced 3D printability and mechanical properties. dECM micro-particles of about 13.4 µm in size were prepared by decellularizing a porcine liver followed by freeze-milling. The new bio-ink, named as dECM powder-based bio-ink (dECM pBio-ink), was prepared by loading the dECM micro-particles into a gelatin mixture. The usefulness of the dECM pBio-ink was evaluated by assessing its mechanical properties, printability, and cytocompatibility. The results showed that its mechanical properties and 3D printability were greatly improved. Its elastic modulus increased by up to 9.17 times that of the conventional dECM bio-ink. Micro-patterns with living cells were successfully achieved with 93% cell viability. Above all, the new bio-ink showed superior performance in stacking of layers for 3D printing, whereas the conventional bio-ink could not maintain its shape. Finally, we demonstrated that the dECM pBio-ink possessed comparable cytocompatibility with the conventional dECM bio-ink through in vitro tests with endothelial cells and primary mouse hepatocytes.

Sequentially induced motor neurons from human fibroblasts facilitate locomotor recovery in a rodent spinal cord injury model.

Generation of autologous human motor neurons holds great promise for cell replacement therapy to treat spinal cord injury (SCI). Direct conversion allows generation of target cells from somatic cells, however, current protocols are not practicable for therapeutic purposes since converted cells are post-mitotic that are not scalable. Therefore, therapeutic effects of directly converted neurons have not been elucidated yet. Here, we show that human fibroblasts can be converted into induced motor neurons (iMNs) by sequentially inducing POU5F1(OCT4) and LHX3. Our strategy enables scalable production of pure iMNs because of the transient acquisition of proliferative iMN-intermediate cell stage which is distinct from neural progenitors. iMNs exhibited hallmarks of spinal motor neurons including transcriptional profiles, electrophysiological property, synaptic activity, and neuromuscular junction formation. Remarkably, transplantation of iMNs showed therapeutic effects, promoting locomotor functional recovery in rodent SCI model. Together, our advanced strategy will provide tools to acquire sufficient human iMNs that may represent a promising cell source for personalized cell therapy.

Oct4 and Hnf4α-induced hepatic stem cells ameliorate chronic liver injury in liver fibrosis model.

Direct conversion from fibroblasts to generate hepatocyte like-cells (iHeps) bypassing the pluripotent state has been described in previous reports as an attractive method acquiring hepatocytes for cell-based therapy. The limited proliferation of iHeps, however, has hampered it uses in cell-based therapy. Since hepatic stem cells (HepSCs) possess self-renewal and bipotency with the capacity to differentiate into both hepatocytes and cholangiocytes, they have therapeutic potential for treating liver disease. Here, we investigated the therapeutic effects of induced HepSCs (iHepSCs) on a carbon tetrachloride (CCl4)-induced liver fibrosis model. We demonstrate that Oct4 and Hnf4a are sufficient to convert fibroblasts into expandable iHepSCs. Hepatocyte-like cells derived from iHepSCs (iHepSC-HEPs) exhibit the typical morphology of hepatocytes and hepatic functions, including glycogen storage, low-density lipoprotein (LDL) uptake, Indocyanine green (ICG) detoxification, drug metabolism, urea production, and albumin secretion. iHepSCs-derived cholangiocyte-like cells (iHepSC-CLCs) expressed cholangiocyte-specific markers and formed cysts and tubule-like structures with apical-basal polarity and secretory function in three-dimensional culture condition. Furthermore, iHepSCs showed anti-inflammatory and anti-fibrotic effects in CCl4-induced liver fibrosis. This study demonstrates that Oct4 and Hnf4α-induced HepSCs show typical hepatic and biliary functionality in vitro. It also presents the therapeutic effect of iHepSCs in liver fibrosis. Therefore, directly converting iHepSCs from somatic cells may facilitate the development of patient-specific cell-based therapy for chronic liver damage.

Regulation of cAMP and GSK3 signaling pathways contributes to the neuronal conversion of glioma.

Glioma is the most malignant type of primary central nervous system tumors, and has an extremely poor prognosis. One potential therapeutic approach is to induce the terminal differentiation of glioma through the forced expression of pro-neural factors. Our goal is to show the proof of concept of the neuronal conversion of C6 glioma through the combined action of small molecules. We investigated the various changes in gene expression, cell-specific marker expression, signaling pathways, physiological characteristics, and morphology in glioma after combination treatment with two small molecules (CHIR99021, a glycogen synthase kinase 3 [GSK3] inhibitor and forskolin, a cyclic adenosine monophosphate [cAMP] activator). Here, we show that the combined action of CHIR99021 and forskolin converted malignant glioma into fully differentiated neurons with no malignant characteristics; inhibited the proliferation of malignant glioma; and significantly down-regulated gene ontology and gene expression profiles related to cell division, gliogenesis, and angiogenesis in small molecule-induced neurons. In vivo, the combined action of CHIR99021 and forskolin markedly delayed neurological deficits and significantly reduced the tumor volume. We suggest that reprogramming technology may be a potential treatment strategy replacing the therapeutic paradigm of traditional treatment of malignant glioma, and a combination molecule comprising a GSK3 inhibitor and a cAMP inducer could be the next generation of anticancer drugs.

Factor-Reduced Human Induced Pluripotent Stem Cells Efficiently Differentiate into Neurons Independent of the Number of Reprogramming Factors.

Reprogramming of somatic cells into induced pluripotent stem cells (iPSCs) by overexpression of the transcription factors OCT4, SOX2, KLF4, and c-Myc holds great promise for the development of personalized cell replacement therapies. In an attempt to minimize the risk of chromosomal disruption and to simplify reprogramming, several studies demonstrated that a reduced set of reprogramming factors is sufficient to generate iPSC. We recently showed that a reduction of reprogramming factors in murine cells not only reduces reprogramming efficiency but also may worsen subsequent differentiation. To prove whether this is also true for human cells, we compared the efficiency of neuronal differentiation of iPSC generated from fetal human neural stem cells with either one (OCT4; hiPSC1F-NSC) or two (OCT4, KLF4; hiPSC2F-NSC) reprogramming factors with iPSC produced from human fibroblasts using three (hiPSC3F-FIB) or four reprogramming factors (hiPSC4F-FIB). After four weeks of coculture with PA6 stromal cells, neuronal differentiation of hiPSC1F-NSC and hiPSC2F-NSC was as efficient as iPSC3F-FIB or iPSC4F-FIB. We conclude that a reduction of reprogramming factors in human cells does reduce reprogramming efficiency but does not alter subsequent differentiation into neural lineages. This is of importance for the development of future application of iPSC in cell replacement therapies.