Mesenchymal stem/stromal cells from human pluripotent stem cell-derived brain organoid enhance the ex vivo expansion and maintenance of hematopoietic stem/progenitor cells
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ABSTRACT:
PROVIDER: PRJNA911317 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR22692861_1.fastq.gz | Fastqsanger.gz | |||
| SRR22692861_2.fastq.gz | Fastqsanger.gz | |||
| SRR22692862_1.fastq.gz | Fastqsanger.gz | |||
| SRR22692862_2.fastq.gz | Fastqsanger.gz | |||
| SRR22692863_1.fastq.gz | Fastqsanger.gz |
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Project description:BackgroundMesenchymal stem/stromal cells (MSCs) are of great therapeutic value due to their role in maintaining the function of hematopoietic stem/progenitor cells (HSPCs). MSCs derived from human pluripotent stem cells represent an ideal alternative because of their unlimited supply. However, the role of MSCs with neural crest origin derived from HPSCs on the maintenance of HSPCs has not been reported.MethodsFlow cytometric analysis, RNA sequencing and differentiation ability were applied to detect the characteristics of stromal cells from 3D human brain organoids. Human umbilical cord blood CD34+ (UCB-CD34+) cells were cultured in different coculture conditions composed of stromal cells and umbilical cord MSCs (UC-MSCs) with or without a cytokine cocktail. The hematopoietic stroma capacity of stromal cells was tested in vitro with the LTC-IC assay and in vivo by cotransplantation of cord blood nucleated cells and stroma cells into immunodeficient mice. RNA and proteomic sequencing were used to detect the role of MSCs on HSPCs.ResultsThe stromal cells, derived from both H1-hESCs and human induced pluripotent stem cells forebrain organoids, were capable of differentiating into the classical mesenchymal-derived cells (osteoblasts, chondrocytes, and adipocytes). These cells expressed MSC markers, thus named pluripotent stem cell-derived MSCs (pMSCs). The pMSCs showed neural crest origin with CD271 expression in the early stage. When human UCB-CD34+ HSPCs were cocultured on UC-MSCs or pMSCs, the latter resulted in robust expansion of UCB-CD34+ HSPCs in long-term culture and efficient maintenance of their transplantability. Comparison by RNA sequencing indicated that coculture of human UCB-CD34+ HSPCs with pMSCs provided an improved microenvironment for HSC maintenance. The pMSCs highly expressed the Wnt signaling inhibitors SFRP1 and SFRP2, indicating that they may help to modulate the cell cycle to promote the maintenance of UCB-CD34+ HSPCs by antagonizing Wnt activation.ConclusionsA novel method for harvesting MSCs with neural crest origin from 3D human brain organoids under serum-free culture conditions was reported. We demonstrate that the pMSCs support human UCB-HSPC expansion in vitro in a long-term culture and the maintenance of their transplantable ability. RNA and proteomic sequencing indicated that pMSCs provided an improved microenvironment for HSC maintenance via mechanisms involving cell-cell contact and secreted factors and suppression of Wnt signaling. This represents a novel method for large-scale production of MSCs of neural crest origin and provides a potential approach for development of human hematopoietic stromal cell therapy for treatment of dyshematopoiesis.
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Project description:We report a novel method for harvesting MSCs from 3D human brain organoids under serum-free culture condition. The stromal cells, derived from both H1-hESCs and human induced pluripotent stem cells (hiPSCs) forebrain organoids, were capable of differentiating into the classical mesenchymal-derived cells (osteoblasts, chondrocytes, and adipocytes). These cells expressed MSC markers, thus named pluripotent stem cell-derived MSCs (pMSCs).To investigate the role of pMSCs, we compared the RNA sequencing of pMSCs ,umbilical cord-MSC,hiPSCs and H1 hESCs.To investigate the function pMSC in the regulation of umbilical cord blood HSPCs, we compared the RNA sequencing of CD34+ cells after co-culturing with and without pMSC and UC-MSCs,respectively.We then performed gene expression profiling analysis using data obtained from RNA-seq of 9 kinds of different cells.
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Project description:Osteolineage cells represent one of the critical bone marrow niche components that support maintenance of hematopoietic stem and progenitor cells (HSPCs). Recent studies demonstrate that extracellular vesicles (EVs) regulate stem cell development via horizontal transfer of bioactive cargo, including microRNAs (miRNAs). Here, we characterize the miRNA profile of EVs secreted by human osteoblasts and study their biological effect of on human umbilical cord blood-derived CD34+ HSPCs by sequencing, gene expression and biochemical analyses. Using next-generation sequencing we show that osteoblast-derived EVs contain highly abundant miRNAs specifically enriched in EVs, including critical regulators of hematopoietic proliferation (e.g., miR-29a). EV treatment of CD34+ HSPCs alters the expression of candidate miRNA targets, such as HBP1, BCL2 and PTEN. Furthermore, EVs enhance proliferation of CD34+ cells and their immature subsets in growth factor-driven ex vivo expansion cultures. Importantly, EV-expanded cells retain their differentiation capacity in vitro and show successful engraftment in NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice in vivo. These discoveries reveal a novel osteoblast-derived EV-mediated mechanism for regulation of HSPC proliferation and warrant consideration of EV-miRNAs for the development of expansion strategies to treat hematological disorders.
2016-10-05 | E-MTAB-4560 | biostudies-arrayexpress
Project description:Direct contact with mesenchymal stromal impacts on migratory behavior and gene expression profile of CD133+ hematopoietic stem cells during ex-vivo expansion Objective: To investigate the impact of direct contact between mesenchymal stromal cells (MSCs) and CD133+ hematopoietic stem cells (HSCs) in terms of expansion potential differentiation, migratory capacity and gene expression profile. Methods: CD133+ purified HSCs were cultured for 7 days on subconfluent MSCs supplemented with growth factor containing medium. After ex-vivo expansion, non-adherent and adherent cells were collected and analyzed separately. Results: The adherent cells were found to have a more immature phenotype compared to the non-adherent fraction. CXCR4 was up regulated in the adherent fraction which was associated with a higher migration capacity towards a SDF-1 gradient. CFU-GM and LTC-IC assays demonstrated a higher clonogenicity and repopulating capacity of the adherent fraction. Genes involved in adhesion, cell cycle control, motility, self-renewal and apoptosis were expressed at a higher level in the adherent fraction. Conclusion: Adhesion and direct cell-cell contact with a MSC feeder layer supports ex-vivo expansion, migratory potential and stemness of CD133+ HSCs. Keywords: co-culture hematopoietic stem cells (HSCs) on mesenchymal stromal cells (MSCs) Non-adherent and adherent fractions from three independent experiments were isolated and collected. Cells were stabilized in PreProtct™ buffer (Miltenyi Biotec, Germany) and stored at -80ºC. Samples were shipped to Miltenyi Biotec (Bergisch Gladbach, Germany) a Whole Human Genome expression analysis. Briefly RNA was extracted and overall quality of total RNA samples was checked via the Agilent 2100 Bioanalyzer platform (Agilent Technologies). RNA samples were amplified and labelled using the Agilent Low RNA Input Linear Amp Kit (Agilent Technologies). Non-adherent samples were pooled as “Non-adherent pool” and adherent samples were pooled as “adherent pool” and labelled with Cy3 and Cy5, respectively. Fluorescence signals of the hybridized Agilent Oligo Microarrays were detected using Agilent’s DNA microarray scanner and the microarray image files were processed with The Agilent Feature Extraction Software (FES).
2008-11-12 | E-GEOD-13566 | biostudies-arrayexpress
Project description:Direct contact with mesenchymal stromal impacts on migratory behavior and gene expression profile of CD133+ hematopoietic stem cells during ex-vivo expansion Objective: To investigate the impact of direct contact between mesenchymal stromal cells (MSCs) and CD133+ hematopoietic stem cells (HSCs) in terms of expansion potential differentiation, migratory capacity and gene expression profile. Methods: CD133+ purified HSCs were cultured for 7 days on subconfluent MSCs supplemented with growth factor containing medium. After ex-vivo expansion, non-adherent and adherent cells were collected and analyzed separately. Results: The adherent cells were found to have a more immature phenotype compared to the non-adherent fraction. CXCR4 was up regulated in the adherent fraction which was associated with a higher migration capacity towards a SDF-1 gradient. CFU-GM and LTC-IC assays demonstrated a higher clonogenicity and repopulating capacity of the adherent fraction. Genes involved in adhesion, cell cycle control, motility, self-renewal and apoptosis were expressed at a higher level in the adherent fraction. Conclusion: Adhesion and direct cell-cell contact with a MSC feeder layer supports ex-vivo expansion, migratory potential and stemness of CD133+ HSCs. Keywords: co-culture hematopoietic stem cells (HSCs) on mesenchymal stromal cells (MSCs)
2008-11-13 | GSE13566 | GEO
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Project description:Among cyclin-dependent kinase inhibitors that control the G1 phase in cell cycle, only p18 and p27 can negatively regulate haematopoietic stem cell (HSC) self-renewal. In this manuscript, we demonstrate that p18 protein is a more potent inhibitor of HSC self-renewal than p27 in mouse models and its deficiency promoted HSC expansion in long-term culture. Single-cell analysis indicated that deleting p18 gene favoured self-renewing division of HSC in vitro. Based on the structure of p18 protein and in-silico screening, we further identified novel smallmolecule inhibitors that can specifically block the activity of p18 protein. Our selected lead compounds were able to expand functional HSCs in a short-term culture. Thus, these putative small-molecule inhibitors for p18 protein are valuable for further dissecting the signalling pathways of stem cell self-renewal and may help develop more effective chemical agents for therapeutic expansion of HSC.
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