Establishment of hematopoietic stem cell compartment after single cell transplantation tracked by FIT approach [scRNA-seq]
Ontology highlight
ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE293145 | GEO | 2025/04/28
REPOSITORIES: GEO
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Project description:This SuperSeries is composed of the SubSeries listed below.
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Project description:Restoration of hematopoiesis upon bone marrow (BM) transplantation relies on the engraftment, expansion and function of transplanted hematopoietic stem cells (HSC). However, the number of donorâderived HSC typically remain much below that present in normal individuals and this likely limits and delays hematopoietic recovery. HSC depend on supportive stromal niches, however, whether the preâconditioning required for BM transplantation damages this stromal niche has not been evaluated extensively. Using mouse models, we now find that that BM stroma cells (BMSC) are severely and permanently damaged by preâconditioning. Transplantation of primary but not cultured BMSC quantitatively reconstitutes stroma function in vivo, which is mediated by a multipotent CD73+ CD105â Sca1+ BMSC subpopulation. BMSC coâtransplantation significantly ameliorates clinical side effects of BM transplantation and doubles expansion of functional, donorâ derived HSC, demonstrating the potential of stroma transplantation to improve HSC transplantation. Purified CD45âTer119âCD73+ CD105â and CD45âTer119âCD73+ CD105+ cells were directly sorted on lysis buffer with FACS Aria 2 in triplicate.
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Project description:Hematopoietic stem cells (HSCs) cultured outside the body are the fundamental component of a wide range of cellular and gene therapies. Recent efforts have achieved more than 200-fold expansion of functional HSCs, but their molecular characterization has not been possible due to the substantial majority of cells in these cultures being non-HSCs and the fact that single cell-initiated cultures display substantial clone-to-clone variability. Using the Fgd5 reporter mouse in combination with EPCR, we have identified an in vitro reporter strategy capable of exclusively identifying HSC-containing clones and prospectively separating HSCs from non-HSCs. Linking single clone functional transplantation data with single clone gene expression profiling, we also reveal the molecular profile of expanded HSCs which is more similar to actively cycling fetal liver HSCs than quiescent adult bone marrow HSCs from which they were derived. A gene expression signature derived from the molecular profiling identifies core molecular components of HSCs, including Prdm16, Fstl1 and Palld, that can further predict the functional outcome in transplantation assays. Taken together, this system represents a new tool to be used for a wide-range of functional screening and molecular experiments previously not possible to carry out due to limited HSC numbers
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Project description:The differentiation capability of induced pluripotent stem cells (iPSCs) towards certain cell types for disease modelling and drug screening assays might be influenced by their somatic cell of origin. Here, we have compared the neural induction of human iPSCs generated from either fetal neural stem cells (fNSCs), dermal fibroblasts or CD34+ hematopoietic stem cells. Neural progenitor cells (NSCs) and neurons could be generated at similar efficiencies from all iPSCs. Whole genome transcriptome analysis and an expression analysis of neural genes in iPSC-derived NSCs revealed a separation of neuroectoderm- from mesoderm-derived cells. Furthermore, we found genes, which were similarly expressed between fNSCs and neuroectoderm- but not mesoderm-derived NSCs. Notably, these neural signatures were retained after transplantation into the cortex of mice and paralleled with increased survival and integration of neuroectoderm-derived cells in vivo. These results indicated distinct origin- dependent neural cell identities in differentiated human iPSCs both in vitro and in vivo. RNA samples for microarray analysis were prepared using RNeasy columns (Qiagen, Germany) with on-column DNA digestion. 300 ng of total RNA per sample were used as the input in the linear amplification protocol (Ambion), which involved the synthesis of T7-linked double-stranded cDNAs and 12hrs of in vitro transcription incorporating biotin-labeled nucleotides. Purified and labeled cRNA was then hybridized for 18 hrs onto HumanHT-12 v4 expression BeadChips (Illumina, USA) following the manufacturer's instructions. After the recommended washing, the chips were stained with streptavidin-Cy3 (GE Healthcare) and scanned using the iScan reader (Illumina) and the accompanying software. The samples were exclusively hybridized as biological replicates. 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