[Show abstract][Hide abstract] ABSTRACT: Epigenetic memory in induced pluripotent stem cells, with regards to their somatic cell type of origin, might lead to variations in their differentiation capacities. In this context, induced pluripotent stem cells from human CD34+ hematopoietic stem cells might be more suitable for hematopoietic differentiation than 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 an epigenetic memory of induced pluripotent stem cells with regards to their somatic cell type of origin, we found a similar hematopoietic induction potential and erythroid differentiation pattern. 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 CD34+ hematopoietic stem cell-derived induced pluripotent stem cells. More detailed methylation analysis of the hematopoietic and erythrocyte promoters identified similar CpG methylation levels in the CD34+-derived and neural stem cell-derived induced pluripotent stem cell lines, which confirms their comparable erythroid differentiation potential.
[Show abstract][Hide abstract] ABSTRACT: The differentiation capability of induced pluripotent stem cells (iPSCs) toward certain cell types for disease modeling and drug screening assays might be influenced by their somatic cell of origin. Here, we have compared the neural induction of human iPSCs generated from fetal neural stem cells (fNSCs), dermal fibroblasts, or cord blood CD34(+) hematopoietic progenitor cells. Neural progenitor cells (NPCs) and neurons could be generated at similar efficiencies from all iPSCs. Transcriptomics analysis of the whole genome and of neural genes revealed a separation of neuroectoderm-derived iPSC-NPCs from mesoderm-derived iPSC-NPCs. Furthermore, we found genes that were similarly expressed in fNSCs and neuroectoderm, but not in mesoderm-derived iPSC-NPCs. Notably, these neural signatures were retained after transplantation into the cortex of mice and paralleled with increased survival of neuroectoderm-derived cells in vivo. These results indicate distinct origin-dependent neural cell identities in differentiated human iPSCs both in vitro and in vivo.
[Show abstract][Hide abstract] ABSTRACT: Surface modification of tissue engineering scaffolds and substrates is required for improving the efficacy of stem cell therapy by generating physicochemical stimulation promoting proliferation and differentiation of stem cells. However, typical surface modification methods including chemical conjugation or physical absorption have several limitations such as multistep, complicated procedures, surface denaturation, batch-to-batch inconsistencies, and low surface conjugation efficiency. In this study, we report a mussel-inspired, biomimetic approach to surface modification for efficient and reliable manipulation of human neural stem cell (NSC) differentiation and proliferation. Our study demonstrates that polydopamine coating facilitates highly efficient, simple immobilization of neurotrophic growth factors and adhesion peptides onto polymer substrates. The growth factor or peptide-immobilized substrates greatly enhance differentiation and proliferation of human NSCs (human fetal brain-derived NSCs and human induced pluripotent stem cell-derived NSCs) at a level comparable or greater than currently available animal-derived coating materials (Matrigel) with safety issues. Therefore, polydopamine-mediated surface modification can provide a versatile platform technology for developing chemically defined, safe, functional substrates and scaffolds for therapeutic applications of human NSCs.
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: 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.
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.
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.
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.
[Show abstract][Hide abstract] ABSTRACT: Reprogramming of human somatic cells represents a valuable tool for enhancing our understanding of the mechanisms underlying
the acquisition of pluripotency and for realizing the potential to generate patient-specific pluripotent stem cells. Induced
pluripotent stem (iPS) cells have been generated from mouse and human somatic cells by the ectopic expression of four transcription
, SOX2, c-MYC, and KLF4). Recently, we reported that Oct4 acting singly is sufficient to directly reprogram adult mouse neural stem cells (NSCs) into iPS cells. Furthermore, we found
that the generation of one-factor (1F) human iPS cells from human NSCs is possible by the ectopic expression of OCT4 alone. These human NSC-derived 1F iPS cells are indistinguishable at the molecular level from human embryonic stem cells
(ESCs) and can differentiate into cells of all three germ lineages both in vitro and in vivo. These findings demonstrate that
the transcription factor OCT4 is sufficient to reprogram mouse and human NSCs into pluripotent cells. This chapter focuses
on the generation of iPS cells from mouse or human NSCs and uses biologic concepts to compare NSCs and pluripotent stem cells
such as ESCs and iPS cells.
KeywordsiPS-Neural stem cells-Reprogramming-OCT4
[Show abstract][Hide abstract] ABSTRACT: 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 TGFβ 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.
[Show abstract][Hide abstract] ABSTRACT: Generation of induced pluripotent stem (iPS) cells from human cord blood (CB)-derived unrestricted somatic stem cells and evaluation of their molecular signature and differentiation potential in comparison to human embryonic stem cells.
Unrestricted somatic stem cells isolated from human CB were reprogrammed to iPS cells using retroviral expression of the transcription factors OCT4, SOX2, KLF4, and C-MYC. The reprogrammed cells were analyzed morphologically, by quantitative reverse transcription polymerase chain reaction, genome-wide microRNA and methylation profiling, and gene expression microarrays, as well as in their pluripotency potential by in vivo teratoma formation in severe combined immunodeficient mice and in vitro differentiation.
CB iPS cells are very similar to human embryonic stem cells morphologically, at their molecular signature, and in their differentiation potential.
Human CB-derived unrestricted somatic stem cells offer an attractive source of cells for generation of iPS cells. Our findings open novel perspectives to generate human leukocyte antigen-matched pluripotent stem cell banks based on existing CB banks. Besides the obvious relevance of a second-generation CB iPS cell bank for pharmacological and toxicological testing, its application for autologous or allogenic regenerative cell transplantation appears feasible.
[Show abstract][Hide abstract] ABSTRACT: Reprogramming of mouse and human somatic cells into induced pluripotent stem (iPS) cells has been possible with retroviral expression of the pluripotency-associated transcription factors Oct4, Sox2, Nanog, and Lin28 as well as Klf4 and c-Myc. iPS cells hold great potential as a model for diseases from the perspective of the individual patient and as an alternative source of pluripotent stem cells for therapeutic applications. In this chapter, we discuss how the use of retroviruses as well as other expression vectors, protein transduction, and small molecules can effectively and efficiently induce pluripotent stem cells from a variety of mouse and human starting somatic cell populations.
Methods in enzymology 01/2010; 476:309-25. DOI:10.1016/S0076-6879(10)76018-3 · 2.19 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: 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.