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PROVIDER: PRJNA771770 | ENA |
REPOSITORIES: ENA
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Project description:Purpose: The population of muscle-derived stem cells called MuStem cells is presented as promising candidate for cell-based therapy of muscle diseases. To validate if this agent can be really presented as therapeutic product and so to be eligible to a future clinical use, it is now required to demonstrate beforehand an efficacy with cells prepared in compliance with good manufacturing practices (GMPs). The aim of the current study was to evaluate the use of two xeno-free blood derivatives corresponding to human serum (HS) and human platelet lysate (hPL) as alternatives to controverted but until now used fetal bovine serum (FBS) for isolation and expansion of human MuStem (hMuStem) cells. Methods: A comparative study was performed with hMuStem cells isolated and in vitro expanded by using commercially available HS and hPL to determine its impact on their proliferation rates, clonogenicity, myogenic commitment level and oligopotency with regard to results obtained under FBS-based medium. Also, their respective phenotype and global gene expression patterns were investigated by flow cytometry and high throughput 3’ digital gene expression RNA-sequencing in order to define a possible differential impact of the human nutrients tested. Results: Comparatively to FBS-based medium, use of HS- and hPL-supplemented ones efficiently supported long-term proliferation of hMuStem cells and enhanced clonogenicity, without main modification of their expression profile and allowing besides limiting the supplementation in growth factors. In vitro differentiation assay combined to transforming growth factor β1 (TGF-β1)-depletion experiments showed a lower myogenic commitment level as well as fusion ability of hMuStem cells when cultured with hPL-based medium according to a TGF-β1-independent process. Use of hPL-derived 3D hydrogel or fibrinogen-depleted hPL demonstrated that heparin-free hPL derivatives maintain consequent myogenic differentiation potential. In addition, the reduced myogenicity was shown to be rapidly reversible following replacement of hPL by HS or fibrinogen-depleted hPL. Conclusions: All together, our original findings position HS and hPL as efficient and suitable alternatives to FBS for preparation of hMuStem cell batch in compliance with GMPs.
2018-05-09 | GSE99085 | GEO
Project description:Donor variation is a prominent critical issue limiting applicability of cell-based therapies. We asked whether reference protocols for pre-clinical bone marrow stromal cell (BMSC) propagation using human platelet lysate (hPL) to replace xenogeneic fetal bovine serum (FBS) might also reduce donor variability for osteo-chondral regeneration. We therefore investigated the impact of donor variability, hPL- vs FBS driven propagation and exhaustive proliferation, on BMSC epigenome, transcriptome, phenotype, coagulation risk and osteochondral regenerative function. Polychromatic flow cytometry revealed maintained canonical fibroblastic phenotype in all cells tested. We confirmed significantly declining proliferative potential in FBS-expanded BMSC after proliferative challenge. Notably, BMSC propagation under the aegis of hPL, compared to FBS, significantly increased BMSC proliferation, created significantly different gene expression trajectories and diverging surface marker signatures, already after just one culture passage. We observed limited but measurable effects on DNA methylation irrespective of culture duration. Moreover, expansion under xenogenic serum conditions resulted in significant loss of function during 3D cartilage organoid-like disk formation and significantly increased clotting risk. Superior chondrogenic function under hPL conditions was maintained over culture duration. As an additional observation, platelet blood group and isoagglutinin levels showed phenotypic changes but only minor impact on BMSC chondrogenesis and clotting behaviour.
2022-11-04 | GSE194298 | GEO
Project description:Donor variation is a prominent critical issue limiting applicability of cell-based therapies. We asked whether reference protocols for pre-clinical bone marrow stromal cell (BMSC) propagation using human platelet lysate (hPL) to replace xenogeneic fetal bovine serum (FBS) might also reduce donor variability for osteo-chondral regeneration. We therefore investigated the impact of donor variability, hPL- vs FBS driven propagation and exhaustive proliferation, on BMSC epigenome, transcriptome, phenotype, coagulation risk and osteochondral regenerative function. Polychromatic flow cytometry revealed maintained canonical fibroblastic phenotype in all cells tested. We confirmed significantly declining proliferative potential in FBS-expanded BMSC after proliferative challenge. Notably, BMSC propagation under the aegis of hPL, compared to FBS, significantly increased BMSC proliferation, created significantly different gene expression trajectories and diverging surface marker signatures, already after just one culture passage. We observed limited but measurable effects on DNA methylation irrespective of culture duration. Moreover, expansion under xenogenic serum conditions resulted in significant loss of function during 3D cartilage organoid-like disk formation and significantly increased clotting risk. Superior chondrogenic function under hPL conditions was maintained over culture duration. As an additional observation, platelet blood group and isoagglutinin levels showed phenotypic changes but only minor impact on BMSC chondrogenesis and clotting behaviour.
2022-11-04 | GSE194295 | GEO
Project description:Proteome characterization of mesenchymal stem cells (MSC) and exosomes.
MSCs were cultured in normoxic, hypoxic and in presence of FBS. Exosomes were prepared from normoxic and hypoxic conditions.
2021-04-27 | PXD025584 | JPOST Repository
Project description:Stem cells from human exfoliated deciduous teeth (SHED) can be used as a potential clinical material. But the use of xenogeneic ingredients, especially fetal bovine serum, will increase the risk of zoonotic disease transmission during the use of animal-derived products. Human-platelet-lysate (hPL) was used in human cell expansion with reliability in clinical applications. This study aimed to explore the feasibility and effect of HPL in the cultivation of SHED. We use proteomics to interpret the impact of HPL on the protein profile of SHED. Propagation, differentiation, qPCR and immunofluorescence were used to detect the effect of HPL on SHED.
2022-05-19 | PXD026687 | Pride
Project description:Recent years have witnessed the introduction of ex vivo expanded dermal fibroblasts for several cell therapy and tissue-engineering applications, including the treatment of facial scars and burns, representing a promising cell type for regenerative medicine. We tested different in-house produced human platelet lysate (HPL) solutions against fetal bovine serum as supplements for in vitro fibroblast expansion by comparing cell yield, molecular marker expression, extracellular matrix (ECM) generation, genomic stability and global gene expression. Our in-house produced HPL supported fibroblast growth at levels similar to those for FBS and commercial HPL products and was superior to AB human serum. Cells grown in HPL maintained a fibroblast phenotype (VIM+, CD44+, CD13+, CD90+), ECM generation capacity (FN+, COL1+) and a normal karyotype, although gene expression profiling revealed changes related to cell metabolism, adhesion and cellular senescence. The HPL manufacturing process was validated within a GMP compliant system and the solution was stable at -80ºC and -20ºC for 2 years. Dermal fibroblasts expanded in vitro with HPL maintain a normal karyotype and expression of fibroblast markers, with only minor changes in their global gene expression profile. Our in-house produced GMP-HPL is an efficient, safe and economical cell culture supplement that can help increase the healthcare activity of blood transfusion centers through the re-use of transfusional plasma and platelets approaching their expiration date. Currently, our HPL solution is approved by the Spanish Agency of Medicines and Medical Devices and is being used in the manufacture of cell therapy products.
2020-10-01 | GSE147146 | GEO
Project description:Purpose: Cyclic mechanical stretch (CMS) promoted proliferation of serum starved mesenchymal stem cells (MSCs) by inhibiting miR-337 expression, similar to FBS. In order to reveal the underlying mechanisms of both CMS and FBS on MSC activation, as well as the regulatory role of miR-337, this research performed high-throughput RNA sequencing (RNA-seq) on MSCs isolated from both wild-type (ET)- and miR-337 knockout- (miR-337-/-) rats. Methods: MSCs isolated form 2-month-old wild-type (WT) and miR-337 knockout (-/-) rats and cultured in vitro to passage 3. After serum starvation for 8h, MSCs were treated with FBS or 10% CMS for another 24h before deep sequencing in triplicate, using Illumina GAIIx. The sequence reads that passed quality filters were analyzed at the transcript isoform level. Results: Using an optimized data analysis workflow, we mapped about 40 million sequence reads per sample. We identified 2472 differentially expressed genes (DEGs) between CMS-stimulated vs. SSt conditions, 2468 DEGs FBS-treated vs. SSt groups. However, only 97 DEGs were identified between CMS and FBS groups, indicating that CMS and FBS groups are more similar to each other and both CMS and FBS groups are quite different from the SSt group. Analysis of DEGs between CMS- or FBS-treated and SSt -treated group revealed that CMS and FBS stimulation activated similar biological processes, of which, PI3K-Akt-mTOR pathway was highly enriched. However, PI3K-Akt-mTOR pathway was not enriched in miR-337-/- MSCs, indicating constitutive activation of this pathway. Conclusions: Our RNA-seq data revealed that both CMS and FBS activates quiescent MSCs through regulating similar genes and pathways. miR-337 is one of the main factors that regulate MSC activation by targeting PI3K-Akt pathway.
2021-12-02 | GSE189845 | GEO
Project description:Induced pluripotent stem cells (iPSCs) can be differentiated toward mesenchymal stromal cells (MSCs), but at least on epigenetic level this transition remains incomplete with the current culture conditions. Hydrogels provide a more physiologic three-dimensional environment for in vitro cell culture than conventional tissue culture plastic (TCP). In this study, we followed the hypothesis that growth and differentiation of primary MSCs and of iPSC-derived MSCs (iMSCs) can be enhanced on hydrogels. To this end, we used a hydrogel made of human platelet lysate (hPL). MSCs were effectively cultured on and inside hPL-gel and demonstrated more structured deposition of extracellular matrix (ECM) components than TCP. Furthermore, hPL-gel supported differentiation of iPSCs toward MSCs. Unexpectedly, the differentiation process seemed to be hardly affected by the substrate: iMSCs generated either on TCP or hPL-gel did not reveal differences in morphology, immunophenotype, or differentiation potential. Moreover, global gene expression and DNA-methylation profiles were almost identical in iMSCs generated on TCP or hPL-gel. Our results indicate that matrix elasticity is less crucial for directed lineage-specific differentiation toward MSCs than expected.
2018-03-13 | GSE95060 | GEO
Project description:Induced pluripotent stem cells (iPSCs) can be differentiated toward mesenchymal stromal cells (MSCs), but at least on epigenetic level this transition remains incomplete with the current culture conditions. Hydrogels provide a more physiologic three-dimensional environment for in vitro cell culture than conventional tissue culture plastic (TCP). In this study, we followed the hypothesis that growth and differentiation of primary MSCs and of iPSC-derived MSCs (iMSCs) can be enhanced on hydrogels. To this end, we used a hydrogel made of human platelet lysate (hPL). MSCs were effectively cultured on and inside hPL-gel and demonstrated more structured deposition of extracellular matrix (ECM) components than TCP. Furthermore, hPL-gel supported differentiation of iPSCs toward MSCs. Unexpectedly, the differentiation process seemed to be hardly affected by the substrate: iMSCs generated either on TCP or hPL-gel did not reveal differences in morphology, immunophenotype, or differentiation potential. Moreover, global gene expression and DNA-methylation profiles were almost identical in iMSCs generated on TCP or hPL-gel. Our results indicate that matrix elasticity is less crucial for directed lineage-specific differentiation toward MSCs than expected.
2018-03-13 | GSE95058 | GEO
Project description:Background: Tissue regeneration and recovery in the adult body depends on self-renewal and differentiation of stem and progenitor cells. Mesenchymal stem cells (MSCs) that have the ability to differentiate into various cell types, have been isolated from the stromal fraction of virtually all tissues. However, little is known about the true identity of MSCs. MSC populations exhibit great tissue-, location- and patient-specific variation in gene expression and are heterogeneous in cell composition. Methodology/Principal findings: Our aim was to analyze the dynamics of differentiation of two closely related stromal cell types, adipose tissue-derived MSCs and dermal fibroblasts (FBs) along adipogenic, osteogenic and chondrogenic lineages using multiplex RNA-seq technology. We found that undifferentiated donor-matched MSCs and FBs are distinct populations that stay different upon differentiation into adipocytes, osteoblasts and chondrocytes. The changes in lineage-specific gene expression occur early in differentiation and persist over time in both MSCs and FBs. Further, MSCs and FBs exhibit similar dynamics of adipogenic and osteogenic differentiation but different dynamics of chondrogenic differentiation. Conclusion: Our findings suggest that stromal stem cells including adipose-derived MSCs and dermal FBs exploit different molecular mechanisms of differentiation to reach a common cell fate. The early mechanisms of differentiation are lineage-specific and are similar for adipogenic and osteogenic differentiation but are distinct for chondrogenic differentiation between MSCs and FBs.
2012-06-28 | GSE37521 | GEO