Efficient Generation of iPS Cells from Skeletal Muscle Stem Cells

Reprogramming of somatic cells into inducible pluripotent stem cells generally occurs at low efficiency, although what limits reprogramming of particular cell types is poorly understood. Recent data suggest that the differentiation status of the cell targeted for reprogramming may influence its susceptibility to reprogramming as well as the differentiation potential of the induced pluripotent stem (iPS) cells that are derived from it. To assess directly the influence of lineage commitment on iPS cell derivation and differentiation, we evaluated reprogramming in adult stem cell and mature cell populations residing in skeletal muscle. Our data using clonal assays and a second-generation inducible reprogramming system indicate that stem cells found in mouse muscle, including resident satellite cells and mesenchymal progenitors, reprogram with significantly greater efficiency than their more differentiated daughters (myoblasts and fibroblasts). However, in contrast to previous reports, we find no evidence of biased differentiation potential among iPS cells derived from myogenically committed cells. These data support the notion that adult stem cells reprogram more efficiently than terminally differentiated cells, and argue against the suggestion that “epigenetic memory” significantly influences the differentiation potential of iPS cells derived from distinct somatic cell lineages in skeletal muscle

Cell Type of Origin Influences the Molecular and Functional Properties of Mouse Induced Pluripotent Stem Cells

Induced pluripotent stem cells (iPSCs) have been derived from various somatic cell populations through ectopic expression of defined factors. It remains unclear whether iPSCs generated from different cell types are molecularly and functionally similar. Here we show that iPSCs obtained from mouse fibroblasts, hematopoietic and myogenic cells exhibit distinct transcriptional and epigenetic patterns. Moreover, we demonstrate that cellular origin influences the in vitro differentiation potentials of iPSCs into embryoid bodies and different hematopoietic cell types. Notably, continuous passaging of iPSCs largely attenuates these differences. Our results suggest that early-passage iPSCs retain a transient epigenetic memory of their somatic cells of origin, which manifests as differential gene expression and altered differentiation capacity. These observations may influence ongoing attempts to use iPSCs for disease modeling and could also be exploited in potential therapeutic applications to enhance differentiation into desired cell lineages.Stem Cell and Regenerative Biolog

Die Rolle des Differenzierungsgrades von somatischen Zellen bei dem Reprogramming in induzierte pluripotente Stammzellen

Introduction 1.1 Mammalian development 1.2 Classification of cells and their developmental potential 1.3 Pluripotent stem cells 1.3.1 ES cells and pluripotency 1.3.2 Pluripotency factors 1.3.3 Other types of pluripotent cells - EC, EG, ES-like and EpiS cell 1.4 Epigenetic regulation in pluripotent cells 1.5 Different strategies to reprogram somatic cells 1.5.1 Reprogramming by nuclear transfer 1.5.2 Reprogramming by cell fusion 1.5.3 Culture-induced reprogramming 1.5.4 “Trans-differentiation” / ”Lineage-conversion” 1.5.5 Direct reprogramming by defined transcription factors into induced pluripotent stem (iPS) cells 1.5.6 Potential clinical and pharmaceutical applications of iPS cells 1.5.7 Development of a “secondary” iPS system 2\. Aims of this thesis 3\. List of publications 3.1 Publication 1: Stem Cells manuscript Eminli et al. 2008 3.2 Publication 2: Nature Genetics manuscript Eminli et al. 2009 3.3 Additional data for Nature Genetics manuscript Eminli et al. 2009 4\. Erklärung über Eigenanteil an den Publikationen 5\. Discussion 5.1 Somatic cells from all three germ layers can be reprogrammed into iPS cells (Aim 1.1 published in Eminli et al. 2008) 5.2 Sox2 is dispensable for the reprogramming of NPCs into iPS cells (Aim 1.2 published in Eminli et al. 2008) 5.3 Differentiation state determines reprogramming potential of hematopoietic cells into iPS cells (Aim 2 published in Eminli et al. 2009) 5.4 Perspectives 6\. Summary 7\. Zusammenfassung 8\. Bibliography 9\. Abbreviation list 10\. Publication record 11\. Acknowledgements-Danksagung 12\. Eidesstattliche ErklärungDirect reprogramming of somatic cells into induced pluripotent stem (iPS) cells has been achieved by overexpression of defined transcription factor combinations such as c-Myc, Klf4, Oct4 and Sox2. iPS cells are molecularly highly similar to embryonic stem (ES) cells and can differentiate into virtually any cell type of the body including germ cells. This observation makes iPS cells very attractive for basic research and potential clinical applications. This PhD thesis consists of two parts; the first part was aimed at studying the role of the somatic cell-of-origin in factor-mediated reprogramming while the second part is aimed at determining whether the differentiation stage of the starting cell affects the efficiency of reprogramming. In the first part, I showed that a defined ectodermal cell type, specifically Neural Progenitor Cells (NPCs), can be reprogrammed into iPS cells, thus demonstrating that cells from all three germ layers are equally amenable to reprogramming into iPS cells by the four factors. Another main conclusion of this part is the observation that cells, which already express one of the four reprogramming factors endogenously, can be reprogrammed with fewer exogenous factors. In particular, NPCs, which express high levels of Sox2, could be reprogrammed into iPS cells in the absence of exogenous Sox2. The second part of this thesis provided the first direct link between the differentiation stage of cells and their reprogramming potential into iPS cells. By using nine different immature hematopoietic cell populations and their differentiated progenies, I showed that immature cells are more amenable to reprogramming than differentiated cell types. For example, adult hematopoietic stem and progenitor cells converted 300 times more efficiently and twice as fast as terminally differentiated cells into iPS cells. Moreover, I was able to translate these observations into a human setting by generating the first human umbilical cord blood-derived iPS cells. In summary, this thesis provides novel biological insights into the role of the somatic cell-of-origin in direct reprogramming and may ultimately facilitate a more efficient and safer generation of patient specific iPS cells.Somatische Zellen können durch die Überexpression von vier verschiedenen Transkriptionsfaktoren, c-Myc, Klf4, Oct4 and Sox2, in sogenannte induzierte pluripotente Stammzellen (iPS Zellen) reprogrammiert werden. Es scheint, dass iPS Zellen und embryonale Stammzellen (ES Zellen) sich in ihren Eigenschaften sehr ähnlich sind. ES Zellen wie auch iPS Zellen haben das Potential, sich in jeden Zelltyp des Körpers zu entwickeln. Diese Eigenschaft macht iPS Zellen zu einem attraktiven Target für die Grundlagenforschung, öffnet aber auch weitreichende Möglichkeiten für potentielle therapeutische Anwendungenszwecke. Diese Doktorarbeit besteht aus zwei Teilen; der erste Teil befasste sich mit der Rolle des Ausgangzelltyps in Bezug auf Faktoren- vermittelte Reprogrammierung während der zweite Teil untersucht, ob der Differenzierungsgrad einen Einfluss auf die Effizienz von somatischer Zellreprogrammierung hat. Im ersten Teil dieser Doktorarbeit konnte gezeigt werden, dass ein bestimmter ektodermaler Zelltyp, insbesondere neurale Vorläuferzellen, in iPS Zellen reprogrammiert werden können, und bestätigte damit, dass Zellabkömmlinge von allen drei Keimblättern gleichermaßen zugänglich sind, um mit den selben 4 Faktoren in iPS Zellen reprogrammiert zu werden. Ein weiteres wichtiges Ergebnis des ersten Projekts ist, dass Zellen, die bereits einen der 4 Reprogrammierungsfaktoren endogen exprimieren, weniger exogene Faktoren benötigen, um iPS Zellen zu produzieren. Es konnte gezeigt werden, dass neurale Vorläuferzellen, die vergleichbare Mengen an Sox2 exprimieren wie ES Zellen, auch ohne virale Überexpression von Sox2 in iPS Zellen reprogrammiert werden können. Im zweiten Teil dieser Doktorarbeit konnte erstmals ein Zusammenhang zwischen dem Differenzierungsgrad somatischer Zellen und deren Reprogrammierungspotential in iPS Zellen aufgezeigt werden. Zu diesem Zweck wurden 9 verschiedene hämatopoetische unreife Zellpopulationen und ihre differenzierten Abkömmlinge isoliert. Weiter konnte gezeigt werden, dass unreifen Zelltypen ein generell viel höheres Reprogrammierungspotential besitzen als differenzierte Zellen. Beispielsweise konnten hämatopoetische Stamm- und Vorläuferzellen 300-fach effizienter und zweimal schneller in iPS Zellen umgewandelt werden verglichen mit differenzierten Zelltypen. Darüber hinaus konnten wir unsere Beobachtungen auch auf humane Zellen übertragen, indem wir zum ersten Mal aufzeigten, dass menschliches Nabelschnurblut in iPS Zellen umgewandelt werden kann. Zusammenfassend lieferte diese Doktorarbeit neuartige biologische Erkenntnisse über die Rolle der somatischen Ausgangsszelle im Zusammenhang mit Faktoren-vermittelter Reprogrammierung und mag damit letztendlich die effiziente und sicherere Erzeugung von Patienten- spezifischen iPS Zellen ermöglichen

Identification and Isolation of Small CD44-Negative Mesenchymal Stem/Progenitor Cells From Human Bone Marrow Using Elutriation and Polychromatic Flow Cytometry

The method of isolation of bone marrow (BM) mesenchymal stem/stromal cells (MSCs) is a limiting factor in their study and therapeutic use. MSCs are typically expanded from BM cells selected on the basis of their adherence to plastic, which results in a heterogeneous population of cells. Prospective identification of the antigenic profile of the MSC population(s) in BM that gives rise to cells with MSC activity in vitro would allow the preparation of very pure populations of MSCs for research or clinical use. To address this issue, we used polychromatic flow cytometry and counterflow centrifugal elutriation to identify a phenotypically distinct population of mesenchymal stem/progenitor cells (MSPCs) within human BM. The MSPC activity resided within a population of rare, small CD45⁻CD73⁺CD90⁺CD105⁺ cells that lack CD44, an antigen that is highly expressed on culture-expanded MSCs. In culture, these MSPCs adhere to plastic, rapidly proliferate, and acquire CD44 expression. They form colony forming units-fibroblast and are able to differentiate into osteoblasts, chondrocytes, and adipocytes under defined in vitro conditions. Their acquired expression of CD44 can be partially downregulated by treatment with recombinant human granulocyte-colony stimulating factor, a response not found in BM-MSCs derived from conventional plastic adherence methods. These observations indicate that MSPCs within human BM are rare, small CD45⁻CD73⁺CD90⁺CD105⁺ cells that lack expression of CD44. These MSPCs give rise to MSCs that have phenotypic and functional properties that are distinct from those of BM-MSCs purified by plastic adherence

Identification and isolation of small CD44-negative mesenchymal stem/progenitor cells from human bone marrow using elutriation and polychromatic flow cytometry

The method of isolation of bone marrow (BM) mesenchymal stem/stromal cells (MSCs) is a limiting factor in their study and therapeutic use. MSCs are typically expanded from BM cells selected on the basis of their adherence to plastic, which results in a heterogeneous population of cells. Prospective identification of the antigenic profile of the MSC population(s) in BM that gives rise to cells with MSC activity in vitro would allow the preparation of very pure populations of MSCs for research or clinical use. To address this issue, we used polychromatic flow cytometry and counterflow centrifugal elutriation to identify a phenotypically distinct population of mesenchymal stem/progenitor cells (MSPCs) within human BM. The MSPC activity resided within a population of rare, small CD45⁻CD73⁺CD90⁺CD105⁺ cells that lack CD44, an antigen that is highly expressed on culture-expanded MSCs. In culture, these MSPCs adhere to plastic, rapidly proliferate, and acquire CD44 expression. They form colony forming units-fibroblast and are able to differentiate into osteoblasts, chondrocytes, and adipocytes under defined in vitro conditions. Their acquired expression of CD44 can be partially downregulated by treatment with recombinant human granulocyte-colony stimulating factor, a response not found in BM-MSCs derived from conventional plastic adherence methods. These observations indicate that MSPCs within human BM are rare, small CD45⁻CD73⁺CD90⁺CD105⁺ cells that lack expression of CD44. These MSPCs give rise to MSCs that have phenotypic and functional properties that are distinct from those of BM-MSCs purified by plastic adherence

Clinically compatible advances in blood-derived endothelial progenitor cell isolation and reprogramming for translational applications

The promise of using induced pluripotent stem cells (iPSCs) for cellular therapies has been hampered by the lack of easily isolatable and well characterized source cells whose genomes have undergone minimal changes during their processing. Blood-derived late-outgrowth endothelial progenitor cells (EPCs) are used for disease modeling and have potential therapeutic uses including cell transplantation and the translation of induced pluripotent stem cell (iPSC) derivatives. However, the current isolation of EPCs has been inconsistent and requires at least 40−80 mL of blood, limiting their wider use. In addition, previous EPC reprogramming methods precluded the translation of EPC-derived iPSCs to the clinic. Here a series of clinically-compatible advances in the isolation and reprogramming of EPCs is presented, including a reduction of blood sampling volumes to 10 mL and use of highly efficient RNA-based reprogramming methods together with autologous human serum, resulting in clinically relevant iPSCs carrying minimal copy number variations (CNVs) compared to their parent line

Wnt Inhibition Facilitates RNA-Mediated Reprogramming of Human Somatic Cells to Naive Pluripotency

In contrast to conventional human pluripotent stem cells (hPSCs) that are related to post-implantation embryo stages, naive hPSCs exhibit features of pre-implantation epiblast. Naive hPSCs are established by resetting conventional hPSCs, or are derived from dissociated embryo inner cell masses. Here we investigate conditions for transgene-free reprogramming of human somatic cells to naive pluripotency. We find that Wnt inhibition promotes RNA-mediated induction of naive pluripotency. We demonstrate application to independent human fibroblast cultures and endothelial progenitor cells. We show that induced naive hPSCs can be clonally expanded with a diploid karyotype and undergo somatic lineage differentiation following formative transition. Induced naive hPSC lines exhibit distinctive surface marker, transcriptome, and methylome properties of naive epiblast identity. This system for efficient, facile, and reliable induction of transgene-free naive hPSCs offers a robust platform, both for delineation of human reprogramming trajectories and for evaluating the attributes of isogenic naive versus conventional hPSCs

Wnt Inhibition Facilitates RNA-Mediated Reprogramming of Human Somatic Cells to Naive Pluripotency.

In contrast to conventional human pluripotent stem cells (hPSCs) that are related to post-implantation embryo stages, naive hPSCs exhibit features of pre-implantation epiblast. Naive hPSCs are established by resetting conventional hPSCs, or are derived from dissociated embryo inner cell masses. Here we investigate conditions for transgene-free reprogramming of human somatic cells to naive pluripotency. We find that Wnt inhibition promotes RNA-mediated induction of naive pluripotency. We demonstrate application to independent human fibroblast cultures and endothelial progenitor cells. We show that induced naive hPSCs can be clonally expanded with a diploid karyotype and undergo somatic lineage differentiation following formative transition. Induced naive hPSC lines exhibit distinctive surface marker, transcriptome, and methylome properties of naive epiblast identity. This system for efficient, facile, and reliable induction of transgene-free naive hPSCs offers a robust platform, both for delineation of human reprogramming trajectories and for evaluating the attributes of isogenic naive versus conventional hPSCs.This research was funded by the Medical Research Council of the United Kingdom (G1001028 and MR/P00072X/1) and European Commission Framework 7 (HEALTHF4-2013-602423, PluriMes). JY was supported by the Guangdong Provincial Key Laboratory, and FvM by a UKRI/MRC Rutherford Fund Fellowship. The Cambridge Stem Cell Institute receives core support from Wellcome and the Medical Research Council. AS is a Medical Research Council Professor

Cell type of origin influences the molecular and functional properties of mouse induced pluripotent stem cells

Induced pluripotent stem cells (iPSCs) have been derived from various somatic cell populations through ectopic expression of defined factors. It remains unclear whether iPSCs generated from different cell types are molecularly and functionally similar. Here we show that iPSCs obtained from mouse fibroblasts, hematopoietic and myogenic cells exhibit distinct transcriptional and epigenetic patterns. Moreover, we demonstrate that cellular origin influences the in vitro differentiation potentials of iPSCs into embryoid bodies and different hematopoietic cell types. Notably, continuous passaging of iPSCs largely attenuates these differences. Our results suggest that early-passage iPSCs retain a transient epigenetic memory of their somatic cells of origin, which manifests as differential gene expression and altered differentiation capacity. These observations may influence ongoing attempts to use iPSCs for disease modeling and could also be exploited in potential therapeutic applications to enhance differentiation into desired cell lineages