Generation of induced pluripotent stem cells from neural stem cells

The generation of induced pluripotent stem (iPS) cells from mouse and human somatic cells by expression of defined transcription factors (Oct4, Sox2, c-Myc, Klf4, Nanog and Lin28) is a powerful tool for conducting basic research and investigating the potential of these cells for replacement therapies. In our laboratory, iPS cells have been generated from adult mouse neural stem cells (NSCs) by ectopic expression of either Oct4 alone (one factor; 1F) or Oct4 plus Klf4 (two factors; 2F). Successful reprogramming of mouse NSCs by 1F or 2F depends on endogenous expression of Sox2, Klf4 and c-Myc. Direct reprogramming of somatic stem cells to 1F or 2F iPS cells avoids expression of the oncogenes Klf4 and c-Myc and, hence, the development of tumors in chimeras and offspring derived from these cells. Here we present a detailed protocol for the derivation of NSCs from adult mouse brain (which takes 4 weeks), and generation of 1F (4-5 weeks) or 2F iPS cells (2-3 weeks) from adult mouse NSCs.close384

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 (LTO-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.close17617

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.close31833

FACS-Assisted CRISPR-Cas9 Genome Editing Facilitates Parkinson's Disease Modeling

Genome editing and human induced pluripotent stem cells hold great promise for the development of isogenic disease models and the correction of disease-associated mutations for isogenic tissue therapy. CRISPR-Cas9 has emerged as a versatile and simple tool for engineering human cells for such purposes. However, the current protocols to derive genome-edited lines require the screening of a great number of clones to obtain one free of random integration or on-locus non-homologous end joining (NHEJ)-containing alleles. Here, we describe an efficient method to derive biallelic genome-edited populations by the use of fluorescent markers. We call this technique FACS-assisted CRISPR-Cas9 editing (FACE). FACE allows the derivation of correctly edited polyclones carrying a positive selection fluorescent module and the exclusion of non-edited, random integrations and on-target allele NHEJ-containing cells. We derived a set of isogenic lines containing Parkinson's-disease-associated mutations in α-synuclein and present their comparative phenotypes

RED BLOOD CELL GENERATION FROM HUMAN INDUCED PLURIPOTENT STEM CELLS OF DIFFERENT DONOR CELL TYPE ORIGIN

Manufacturing red blood cells (RBC) from human induced pluripotent stem cells (iPS cells) offers the potential to produce large quantities of patients' specific RBC for transfusions purposes. Epigenetic memory in iPS cells in regard to their donor cell type of origin might lead to variations in their differentiation capacities. We have generated iPS cells from human cord blood CD34+ hematopoietic stem cells (HSC) (CD34-iPS) and neural stem cells (NSC-iPS) and evaluated their differentiation potential into hematopoietic precursor and mature RBC. For hematopoietic induction, iPS cells were allowed to form embryoid bodies (EBs) under cytokine stimulation for 21 days. Thereafter, dissociated single cells were applied to a three-step protocol for human erythropoiesis for additional 18-25 days. We have found a similar hematopoietic induction potential among our cell lines. After EB dissociation on day 21, hematopoietic commitment, measured by CD43 expression, was about 20% for all cell lines. Colony-forming unit assays demonstrate a similar distribution of myeloid (CFU-M/CFU-GM), erythroid (BFU-E/CFU-E) and mixed (CFU-GEMM) colonies. Hematopoietic cells further developed into erythroid precursors as determined by >90% expression of glycophorin A, followed by maturation into normoblasts and partially enucleated RBC. All human iPS derived erythrocytes predominantly present fetal hemoglobin (>85%), some embryonic and only a minor amount of adult hemoglobin. In summary, we were able to recapitulate the development of RBC from all human iPS cell lines evaluated. In addition, our data hint at a similar erythrocyte induction potential of iPS cells, independent of their donor cell type origin

Oct4 and Hnf4 alpha-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 Hnf4 alpha-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

Induced Neural Stem Cells Achieve Long-Term Survival and Functional Integration in the Adult Mouse Brain

SummaryDifferentiated cells can be converted directly into multipotent neural stem cells (i.e., induced neural stem cells [iNSCs]). iNSCs offer an attractive alternative to induced pluripotent stem cell (iPSC) technology with regard to regenerative therapies. Here, we show an in vivo long-term analysis of transplanted iNSCs in the adult mouse brain. iNSCs showed sound in vivo long-term survival rates without graft overgrowths. The cells displayed a neural multilineage potential with a clear bias toward astrocytes and a permanent downregulation of progenitor and cell-cycle markers, indicating that iNSCs are not predisposed to tumor formation. Furthermore, the formation of synaptic connections as well as neuronal and glial electrophysiological properties demonstrated that differentiated iNSCs migrated, functionally integrated, and interacted with the existing neuronal circuitry. We conclude that iNSC long-term transplantation is a safe procedure; moreover, it might represent an interesting tool for future personalized regenerative applications

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.close131

Stepwise Clearance of Repressive Roadblocks Drives Cardiac Induction in Human ESCs

Cardiac induction requires stepwise integration of BMP and WNT pathway activity. Human embryonic stem cells (hESCs) are developmentally and clinically relevant for studying the poorly understood molecular mechanisms downstream of these cascades. We show that BMP and WNT signaling drive cardiac specification by removing sequential roadblocks that otherwise redirect hESC differentiation toward competing fates, rather than activating a cardiac program per se. First, BMP and WNT signals pattern mesendoderm through cooperative repression of SOX2, a potent mesoderm antagonist. BMP signaling promotes miRNA-877 maturation to induce SOX2 mRNA degradation, while WNT-driven EOMES induction transcriptionally represses SOX2. Following mesoderm formation, cardiac differentiation requires inhibition of WNT activity. We found that WNT inhibition serves to restrict expression of anti-cardiac regulators MSX1 and CDX2/1. Conversely, their simultaneous disruption partially abrogates the requirement for WNT inactivation. These results suggest that human cardiac induction depends on multi-stage repression of alternate lineages, with implications for deriving expandable cardiac stem cells

Induction of Pluripotency in Adult Unipotent Germline Stem Cells

Mouse and human stem cells with features similar to those of embryonic stem cells have been derived from testicular cells. Although pluripotent stem cells have been obtained from defined germline stem cells (GSCs) of mouse neonatal testis, only multipotent stem cells have been obtained so far from defined cells of mouse adult testis. In this study we describe a robust and reproducible protocol for obtaining germline-derived pluripotent stem (gPS) cells from adult unipotent GSCs. Pluripotency of gPS cells was confirmed by in vitro and in vivo differentiation, including germ cell contribution and transmission. As determined by clonal analyses, gPS cells indeed originate from unipotent GSCs. We propose that the conversion process requires a GSC culture microenvironment that depends on the initial number of plated GSCs and the length of culture time.close11812