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Human serum and platelet lysate are appropriate xeno-free alternatives for clinical-grade production of human MuStem cell batches.

ABSTRACT: BACKGROUND:Canine MuStem cells have demonstrated regenerative efficacy in a dog model of muscular dystrophy, and the recent characterization of human counterparts (hMuStem) has highlighted the therapeutic potential of this muscle-derived stem cell population. To date, these cells have only been generated in research-grade conditions. However, evaluation of the clinical efficacy of any such therapy will require the production of hMuStem cells in compliance with good manufacturing practices (GMPs). Because the current use of fetal bovine serum (FBS) to isolate and expand hMuStem cells raises several ethical, safety, and supply concerns, we assessed the use of two alternative xeno-free blood derivatives: human serum (HS) and a human platelet lysate (hPL). METHODS:hMuStem cells were isolated and expanded in vitro in either HS-supplemented or hPL-supplemented media and the proliferation rate, clonogenicity, myogenic commitment potential, and oligopotency compared with that observed in FBS-supplemented medium. Flow cytometry and high-throughput 3'-digital gene expression RNA sequencing were used to characterize the phenotype and global gene expression pattern of hMuStem cells cultured with HS or hPL. RESULTS:HS-supplemented and hPL-supplemented media both supported the isolation and long-term proliferation of hMuStem cells. Compared with FBS-based medium, both supplements enhanced clonogenicity and allowed for a reduction in growth factor supplementation. Neither supplement altered the cell lineage pattern of hMuStem cells. In vitro differentiation assays revealed a decrease in myogenic commitment and in the fusion ability of hMuStem cells when cultured with hPL. In return, this reduction of myogenic potential in hPL-supplemented cultures was rapidly reversed by substitution of hPL with HS or fibrinogen-depleted hPL. Moreover, culture of hMuStem cells in hPL hydrogel and fibrinogen-depleted hPL demonstrated that myogenic differentiation potential is maintained in heparin-free hPL derivatives. CONCLUSIONS:Our findings indicate that HS and hPL are efficient and viable alternatives to FBS for the preparation of hMuStem cell batches in compliance with GMPs.

SUBMITTER: Saury C

PROVIDER: S-EPMC5932844 | biostudies-literature | 2018 May

REPOSITORIES: biostudies-literature

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Human serum and platelet lysate are appropriate xeno-free alternatives for clinical-grade production of human MuStem cell batches.

Saury Charlotte C Lardenois Aurélie A Schleder Cindy C Leroux Isabelle I Lieubeau Blandine B David Laurent L Charrier Marine M Guével Laëtitia L Viau Sabrina S Delorme Bruno B Rouger Karl K

Stem cell research & therapy 20180502 1

<h4>Background</h4>Canine MuStem cells have demonstrated regenerative efficacy in a dog model of muscular dystrophy, and the recent characterization of human counterparts (hMuStem) has highlighted the therapeutic potential of this muscle-derived stem cell population. To date, these cells have only been generated in research-grade conditions. However, evaluation of the clinical efficacy of any such therapy will require the production of hMuStem cells in compliance with good manufacturing practice ...[more]

PMID: 29720259

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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.

Project description:Mesenchymal stromal cells (MSCs) are non-hematopoietic cells that have a broad therapeutic potential. To obtain sufficient cells for clinical application, they must be expanded ex vivo. In the initial expansion protocols described, fetal calf serum (FCS) was used as the reference growth supplement, but more recently different groups started to replace FCS with platelet lysate (PL). We investigated in this study the impact of the culture supplement on gene expression of MSCs. Human bone marrow derived MSCs were expanded in vitro in FCS and PL supplemented medium. We found that MSCs expanded in PL-containing medium (PL-MSCs) express typical MSC immunomorphological features and can migrate, as their counterparts expanded in FCS-containing medium, through a layer of endothelial cells in vitro. Additionally, they show an increased proliferation rate compared to MSCs expanded in FCS medium (FCS-MSCs). RNA sequencing performed for MSCs cultured in both types of expansion medium revealed a large impact of the choice of growth supplement on gene expression: 1974 genes were at least twofold up- or downregulated. We focused on impact of genes involved in apoptosis and senescence. Our data showed that PL-MSCs express more anti-apoptotic genes and FCS-MSCs more pro-apoptotic genes. FCS-MSCs showed upregulation of senescence-related genes after four passages whereas this was rarer in PL-MSCs at the same timepoint. Since PL-MSCs show higher proliferation rates and anti-apoptotic gene expression, they might acquire features that predispose them to malignant transformation. We screened 10 MSC samples expanded in PL-based medium for the presence of tumor-associated genetic variants using a 165 gene panel and detected only 21 different genetic variants. According to our analysis, none of these were established pathogenic mutations. Our data show that differences in culture conditions such as growth supplement have a significant impact on the gene expression profile of MSCs and favor the use of PL over FCS for expansion of MSCs.

Project description:In vitro two-dimensional (2D) and three-dimensional (3D) cultivation of mammalian cells requires supplementation with serum. Mesenchymal stem cells (MSCs) are widely used in clinical trials for bioregenerative medicine and in most cases, in vitro expansion and differentiation of these cells are required before application. Optimized expansion and differentiation protocols play a key role in the treatment outcome. 3D cell cultivation systems are more comparable to in vivo conditions and can provide both, more physiological MSC expansion and a better understanding of intercellular and cell-matrix interactions. Xeno-free cultivation conditions minimize risks of immune response after implantation. Human platelet lysate (hPL) appears to be a valuable alternative to widely used fetal calf serum (FCS) since no ethical issues are associated with its harvest, it contains a high concentration of growth factors and cytokines and it can be produced from expired platelet concentrate. In this study, we analyzed and compared proliferation, as well as osteogenic and chondrogenic differentiation of human adipose tissue-derived MSCs (hAD-MSC) using three different supplements: FCS, human serum (HS), and hPL in 2D. Furthermore, online monitoring of osteogenic differentiation under the influence of different supplements was performed in 2D. hPL-cultivated MSCs exhibited a higher proliferation and differentiation rate compared to HS- or FCS-cultivated cells. We demonstrated a fast and successful chondrogenic differentiation in the 2D system with the addition of hPL. Additionally, FCS, HS, and hPL were used to formulate Gelatin-methacryloyl (GelMA) hydrogels in order to evaluate the influence of the different supplements on the cell spreading and proliferation of cells growing in 3D culture. In addition, the hydrogel constructs were cultivated in media supplemented with three different supplements. In comparison to FCS and HS, the addition of hPL to GelMA hydrogels during the encapsulation of hAD-MSCs resulted in enhanced cell spreading and proliferation. This effect was promoted even further by cultivating the hydrogel constructs in hPL-supplemented media.

Dataset Information

Human serum and platelet lysate are appropriate xeno-free alternatives for clinical-grade production of human MuStem cell batches.

Publications

Human serum and platelet lysate are appropriate xeno-free alternatives for clinical-grade production of human MuStem cell batches.

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