Dataset Information

Stimulation of Activin A/Nodal signaling is insufficient to induce definitive endoderm formation of cord blood-derived unrestricted somatic stem cells.

ABSTRACT:

Introduction

Unrestricted somatic stem cells (USSC) derived from umbilical cord blood are an attractive alternative to human embryonic stem cells (hESC) for cellular therapy. USSC are capable of forming cells representative of all three germ line layers. The aim of this study was to determine the potential of USSC to form definitive endoderm following induction with Activin A, a protein known to specify definitive endoderm formation of hESC.

Methods

USSC were cultured for (1) three days with or without 100 ng/ml Activin A in either serum-free, low-serum or serum-containing media, (2) three days with or without 100 ng/ml Activin A in combination with 10 ng/ml FGF4 in pre-induction medium, or (3) four days with or without small molecules Induce Definitive Endoderm (IDE1, 100 nM; IDE2, 200 nM) in serum-free media. Formation of definitive endoderm was assessed using RT-PCR for gene markers of endoderm (Sox17, FOXA2 and TTF1) and lung epithelium (surfactant protein C; SPC) and cystic fibrosis transmembrane conductance regulator; CFTR). The differentiation capacity of Activin A treated USSC was also assessed.

Results

Activin A or IDE1/2 induced formation of Sox17+ definitive endoderm from hESC but not from USSC. Activin A treated USSC retained their capacity to form cells of the ectoderm (nerve), mesoderm (bone) and endoderm (lung). Activin A in combination with FGF4 did not induce formation of Sox17+ definitive endoderm from USSC. USSC express both Activin A receptor subunits at the mRNA and protein level, indicating that these cells are capable of binding Activin A.

Conclusions

Stimulation of the Nodal signaling pathway with Activin A or IDE1/2 is insufficient to induce definitive endoderm formation from USSC, indicating that USSC differ in their stem cell potential from hESC.

SUBMITTER: Filby CE

PROVIDER: S-EPMC3226287 | biostudies-literature | 2011 Apr

REPOSITORIES: biostudies-literature

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Publications

Stimulation of Activin A/Nodal signaling is insufficient to induce definitive endoderm formation of cord blood-derived unrestricted somatic stem cells.

Filby Caitlin E CE Williamson Robert R van Kooy Peter P Pébay Alice A Dottori Mirella M Elwood Ngaire J NJ Zaibak Faten F

Stem cell research & therapy 20110404 2

<h4>Introduction</h4>Unrestricted somatic stem cells (USSC) derived from umbilical cord blood are an attractive alternative to human embryonic stem cells (hESC) for cellular therapy. USSC are capable of forming cells representative of all three germ line layers. The aim of this study was to determine the potential of USSC to form definitive endoderm following induction with Activin A, a protein known to specify definitive endoderm formation of hESC.<h4>Methods</h4>USSC were cultured for (1) thre ...[more]

PMID: 21463501

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Project description:Induced pluripotent stem (iPS) cells have been generated from mouse and human somatic cells by ectopic expression of the transcription factors OCT4, SOX2, KLF4, c-MYC as well as NANOG and LIN28. Here we report generation of induced pluripotent stem cells from human umbilical cord blood derived unrestricted somatic stem cells (USSC) using retroviral expression of the transcription factors OCT4, SOX2, KLF4 and C-MYC and evaluation of their molecular signature and differentiation potential in comparison to human embryonic stem cells. The reprogrammed cells (HUiPS) were analysed morphologically, by qRT-PCR, global miRNA and epigenetic profiling and gene expression microarrays, as well as in their in vitro and in vivo differentiation potential by embryoid body formation and teratoma assay. The cord blood iPS cells are highly similar to human embryonic stem cells morphologically, at their molecular signature as well as in their in vitro and in vivo differentiation potential. Human cord blood derived unrestricted somatic stem cells offer an attractive source of cells for the generation of induced pluripotent stem cells. Our findings open novel perspectives to generate HLA matched pluripotent stem cell banks based on existing cord blood banks. Besides its obvious relevance of such a second generation cord blood iPS bank for pharmacological and toxicological testing, its application for autologous or allogenic regenerative cell transplantation appears feasible. For transcriptome profiling, 400 ng of total DNA-free RNA was used as input for labelled cRNA synthesis (Illumina TotalPrep RNA Amplification Kit - Ambion) following the manufacturer's instructions (IVT: 10h). Quality-checked cRNA samples were hybridized as biological or technical duplicates for 18 h onto HumanRef-8 v3 expression BeadChips (Illumina), washed, stained, and scanned following guidelines and using materials / instrumentation supplied / suggested by the manufacturer. Six sample types were analyzed, each one of them in duplicate. USSC: human umbilical cord blood unrestricted somatic stem cells (duplicates) HUiPS: human iPS cells from human umbilical cord blood USSC, hand-picked cols (duplicates) H9 hESC: H9 human ESCs grown on low-density CF1 MEFs (duplicates) H1 hESC: H1 human ESCs grown on low-density CF1 MEFs (duplicates) 1F hNiPS: One factor (Oct4) human iPS cells from hNSCs, hand-picked cols (duplicates) 2F hNiPS: Two factors (Oct4, Klf4) human iPS cells from hNSCs, hand-picked cols (duplicates)

Dataset Information

Stimulation of Activin A/Nodal signaling is insufficient to induce definitive endoderm formation of cord blood-derived unrestricted somatic stem cells.

Introduction

Methods

Results

Conclusions

Publications

Stimulation of Activin A/Nodal signaling is insufficient to induce definitive endoderm formation of cord blood-derived unrestricted somatic stem cells.

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