17 research outputs found
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 âepigenetic memoryâ significantly influences the differentiation potential of iPS cells derived from distinct somatic cell lineages in skeletal muscle
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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 âTrans-differentiationâ / âLineage-conversionâ 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 âsecondaryâ 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\.
ErklĂ€rung uÌber 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
ErklÀrungDirect 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 Ăberexpression 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 Àhnlich 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 fuÌr die Grundlagenforschung, öffnet aber auch
weitreichende Möglichkeiten fuÌr 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 wÀhrend 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
VorlÀuferzellen, in iPS Zellen reprogrammiert werden können, und bestÀtigte
damit, dass Zellabkömmlinge von allen drei KeimblĂ€ttern gleichermaĂen
zugÀnglich 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 VorlÀuferzellen, die vergleichbare Mengen an Sox2
exprimieren wie ES Zellen, auch ohne virale Ăberexpression 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 hÀmatopoetische 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 hÀmatopoetische
Stamm- und VorlÀuferzellen 300-fach effizienter und zweimal schneller in iPS
Zellen umgewandelt werden verglichen mit differenzierten Zelltypen. DaruÌber
hinaus konnten wir unsere Beobachtungen auch auf humane Zellen uÌbertragen,
indem wir zum ersten Mal aufzeigten, dass menschliches Nabelschnurblut in iPS
Zellen umgewandelt werden kann. Zusammenfassend lieferte diese Doktorarbeit
neuartige biologische Erkenntnisse uÌber 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 CD45â»CD73âșCD90âșCD105âș 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 CD45â»CD73âșCD90âșCD105âș 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 CD45â»CD73âșCD90âșCD105âș 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 CD45â»CD73âșCD90âșCD105âș 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 40â80 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