Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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Re-specification of myeloid precursors from human pluripotent cells into engraftable multi-lineage progenitors

ABSTRACT: Human pluripotent stem cells were differentiated into hematopoietic progenitors, which were then re-specified using defined transcription factors to resemble hematopoietic stem cells (HSC) We used microarrays to establish the similarity between converted cells and purified human HSCs. The samples analyzed were: starting embryoid body progenitors, transcription factor-converted cells, and primary HSCs and progenitors from fetal liver and cord blood. All samples were flow sorted for CD34+ and CD38- to compare across a similar population of primitive cells.

ORGANISM(S): Homo sapiens

SUBMITTER: Sergei Doulatov

PROVIDER: E-GEOD-49938 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Although a considerable number of reports indicate an involvement of the Hox-A10 gene in the molecular control of hematopoiesis, the conclusions of such studies are quite controversial since they support, in some cases, a role in the stimulation of stem cell self-renewal and myeloid progenitor expansion while, in others, implicate this transcription factor in the induction of monocyte - macrophage differentiation. To clarify this issue we analyzed the biological effects and the transcriptome changes determined in human primary CD34+ hematopoietic progenitors by retroviral transduction of a full length Hox-A10 cDNA. The results obtained clearly indicated that this homeogene is an inducer of monocyte differentiation, at least partly acting through the up-regulation of MafB gene, recently identified as master regulator of such maturation pathway. By using a combined approach based on computational analysis, EMSA experiments and luciferase assays, we were able to demonstrate the presence of a Hox-A10 binding site in the promoter region of the MafB gene, which suggested the likely molecular mechanism underlying the observed effect. Interestingly, stimulation of the same cells with the Vitamin D3 monocyte differentiation inducer resulted in a clear increase of Hox-A10 and MafB transcripts, indicating the existence of a precise transactivation cascade involving VDR, Hox-A10 and MafB transcription factors. Altogether these data allow to conclude that the Vitamin D3 / Hox-A10 pathway supports MafB function during the induction of monocyte differentiation. Experiment Overall Design: RNA pools (100 ng) of LXIDN and LHoxA10IDN transduced CD34+ cells, obtained from three independent experiments, were converted in labelled cRNA according with the âTwo cycleâ protocol advised by Affymetrix. cRNA has been used to hybridise Affymetrix HG-U133A GeneChip arrays. Images obtained by scanning chips of LXIDN and LHoxA10IDN transduced CD34+ cells were processed using the GeneChip Operating Software. Microarray analysis of Hox-A10 transduced CD34+ cells provided a substantial contribute for a better comprehension of the biological effects driven by this transcription factor in human primary hematopoietic stem / progenitor cells. Results of this analysis confirmed the stimulatory effect exerted by Hox-A10 on monocytopoiesis, disclosing an up-regulated expression of transcription factors and differentiation markers (CD antigens, granule proteins, cytokines / chemokines) that are typically associated with this maturation lineage. In addition, they also evidenced a decreased expression of genes related to erythroid and granulocyte differentiation programs. This last effect was also confirmed by cytochemical and morphological evaluation of Hox-A10 transduced CD34+ cells.

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Re-specification of myeloid precursors from human pluripotent cells into engraftable multi-lineage progenitors

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