Upregulation of mitotic bookmarking factors during enhanced proliferation of human stromal cells in human platelet lysate.
ABSTRACT: BACKGROUND:Innovative human stromal cell therapeutics require xeno-free culture conditions. Various formulations of human platelet lysate (HPL) are efficient alternatives for fetal bovine serum (FBS). However, a consistent lack of standardized manufacturing protocols and quality criteria hampers comparability of HPL-products. Aim of this study was to compare the biochemical composition of three differential HPL-preparations with FBS and to investigate their impact on stromal cell biology. METHODS:Stromal cells were isolated from bone marrow (BM), white adipose tissue (WAT) and umbilical cord (UC) and cultured in medium supplemented with pooled HPL (pHPL), fibrinogen-depleted serum-converted pHPL (pHPLS), mechanically fibrinogen-depleted pHPL (mcpHPL) and FBS. Biochemical parameters were analyzed in comparison to standard values in whole blood. Distinct growth factors and cytokines were measured by bead-based multiplex technology. Flow cytometry of stromal cell immunophenotype, in vitro differentiation, and mRNA expression analysis of transcription factors SOX2, KLF4, cMYC, OCT4 and NANOG were performed. RESULTS:Biochemical parameters were comparable in all pHPL preparations, but to some extent different to FBS. Total protein, glucose, cholesterol and Na+ were elevated in pHPL preparations, K+ and Fe3+ levels were higher in FBS. Compared to FBS, pHPL-based media significantly enhanced stromal cell propagation. Characteristic immunophenotype and in vitro differentiation potential were maintained in all four culture conditions. The analysis of growth factors and cytokines revealed distinct levels depending on the pre-existence in pHPL, consumption or secretion by the stromal cells. Interestingly, mRNA expression of the transcription and mitotic bookmarking factors cMYC and KLF4 was significantly enhanced in a source dependent manner in stromal cells cultured in pHPL- compared to FBS-supplemented media. SOX2 mRNA expression of all stromal cell types was increased in all pHPL culture conditions. CONCLUSION:All pHPL-supplemented media equally supported proliferation of WAT- and UC-derived stromal cells significantly better than FBS. Mitotic bookmarking factors, known to enable a quick re-entry to the cell cycle, were significantly enhanced in pHPL-expanded cells. Our results support a better characterization and standardization of humanized culture media for stromal cell-based medicinal products.
Project description:Human platelet lysate (hPL) has been considered as the preferred supplement for the xeno-free stem cell culture for many years. However, the biological effect of hPL on the proliferation and differentiation of dental stem cells combined with the use of medical grade synthetic biomaterial is still under investigation. Thus, the optimal scaffold composition, cell type and specific growth conditions, yet need to be formulated. In this study, we aimed to investigate the regenerative potential of dental stem cells seeded on synthetic scaffolds and maintained in osteogenic media supplemented with either hPL or xeno-derived fetal bovine serum (FBS). Two types of dental stem cells were isolated from human impacted third molars and intact teeth; stem cells of apical papilla (SCAP) and periodontal ligament stem cells (PDLSCs). Cells were expanded in cell culture media supplemented with either hPL or FBS. Consequently, proliferative capacity, immunophenotypic characteristics and multilineage differentiation potential of the derived cells were evaluated on monolayer culture (2D) and on synthetic scaffolds fabricated from poly 'lactic-co-glycolic' acid (PLGA) (3D). The functionality of the induced cells was examined by measuring the concentration of osteogenic markers ALP, OCN and OPN at different time points. Our results indicate that the isolated dental stem cells showed similar mesenchymal characteristics when cultured on hPL or FBS-containing culture media. Scanning electron microscopy (SEM) and H&E staining revealed the proper adherence of the derived cells on the 3D scaffold cultures. Moreover, the increase in the concentration of osteogenic markers proved that hPL was able to produce functional osteoblasts in both culture conditions (2D and 3D), in a way similar to FBS culture. These results reveal that hPL provides a suitable substitute to the animal-derived serum, for the growth and functionality of both SCAP and PDLSCs. Thus the use of hPL, in combination with PLGA scaffolds, can be useful in future clinical trials for dental regeneration.
Project description:Due to their immunosuppressive properties, mesenchymal stem cells (MSC) have been evaluated for the treatment of immunological diseases. However, the animal-derived growth supplements utilized for MSC manufacturing may lead to clinical complications. Characterization of alternative media formulations is imperative for MSC therapeutic application. Human BMMSC and AdMSC were expanded in media supplemented with either human platelet lysates (HPL), serum-free media/xeno-free FDA-approved culture medium (SFM/XF), or fetal bovine serum (FBS) and the effects on their properties were investigated. The immunophenotype of resting and IFN-? primed BMMSC and AdMSC remained unaltered in all media. Both HPL and SFM/XF increased the proliferation of BMMSC and AdMSC. Expansion of BMMSC and AdMSC in HPL increased their differentiation, compared to SFM/XF and FBS. Resting BMMSC and AdMSC, expanded in FBS or SFM/XF, demonstrated potent immunosuppressive properties in both non-primed and IFN-? primed conditions, whereas HPL-expanded MSC exhibited diminished immunosuppressive properties. Finally, IFN-? primed BMMSC and AdMSC expanded in SFM/XF and HPL expressed attenuated levels of IDO-1 compared to FBS. Herein, we provide strong evidence supporting the use of the FDA-approved SFM/XF medium, in contrast to the HPL medium, for the expansion of MSC towards therapeutic applications.
Project description:Human embryonic stem cell-derived mesenchymal progenitor (hES-MP) cells are mesenchymal-like cells, derived from human embryonic stem cells without the aid of feeder cells. They have been suggested as a potential alternative to mesenchymal stromal cells (MSCs) in regenerative medicine due to their mesenchymal-like proliferation and differentiation characteristics. Cells and cell products intended for regenerative medicine in humans should be derived, expanded and differentiated using conditions free of animal-derived products to minimize risk of animal-transmitted disease and immune reactions to foreign proteins. Human platelets are rich in growth factors needed for cell culture and have been used successfully as an animal serum replacement for MSC expansion and differentiation. In this study, we compared the proliferation of hES-MP cells and MSCs; the hES-MP cell growth was sustained for longer than that of MSCs. Growth factors, gene expression, and surface marker expression in hES-MP cells cultured with either human platelet lysate (hPL) or fetal bovine serum (FBS) supplementation were compared, along with differentiation to osteogenic and chondrogenic lineages. Despite some differences between hES-MP cells grown in hPL- and FBS-supplemented media, hPL was found to be a suitable replacement for FBS. In this paper, we demonstrate for the first time that hES-MP cells can be grown using platelet lysates from expired platelet concentrates (hPL).
Project description:BACKGROUND:For adipose-derived stromal cells (ASCs) to be safe for use in the clinical setting, they need to be prepared using good manufacturing practices (GMPs). Fetal bovine serum (FBS), used to expand ASCs in vitro in some human clinical trials, runs the risk of xenoimmunization and zoonotic disease transmission. To ensure that GMP standards are maintained, pooled human platelet lysate (pHPL) has been used as an alternative to FBS. ASCs proliferate more rapidly in pHPL than in FBS, with no significant change in immunophenotype and differentiation capacity. However, not much is known about how pHPL affects the transcriptome of these cells. METHODS:This study investigated the effect of pHPL and FBS on the ASC transcriptome during in vitro serial expansion from passage 0 to passage 5 (P0 to P5). RNA was isolated from ASCs at each passage and hybridized to Affymetrix HuGene 2.0 ST arrays for gene expression analysis. RESULTS:We observed that the transcriptome of ASCs expanded in pHPL (pHPL-ASCs) and FBS (FBS-ASCs) had the greatest change in gene expression at P2. Gene ontology revealed that genes upregulated in pHPL-ASCs were enriched for cell cycle, migration, motility, and cell-cell interaction processes, while those in FBS-ASCs were enriched for immune response processes. ASC transcriptomes were most homogenous from P2 to P5 in FBS and from P3 to P5 in pHPL. FBS- and pHPL-gene-specific signatures were observed, which could be used as markers to identify cells previously grown in either FBS or pHPL for downstream clinical/research applications. The number of genes constituting the FBS-specific effect was 3 times greater than for pHPL, suggesting that pHPL may be a milder supplement for cell expansion. A set of genes were expressed in ASCs at all passages and in both media. This suggests that a unique ASC in vitro transcriptomic profile exists that is independent of the passage number or medium used. CONCLUSIONS:GO classification revealed that pHPL-ASCs are more involved in cell cycle processes and cellular proliferation when compared to FBS-ASCs, which are involved in more specialized or differentiation processes like cardiovascular and vascular development. This makes pHPL a potential superior supplement for expanding ASCs as they retain their proliferative capacity, remain untransformed and pHPL does not affect the genes involved in differentiation in specific developmental processes.
Project description:Stem cells derived from the subcutaneous adipose tissue of debrided burned skin represent an appealing source of adipose-derived stem cells (ASCs) for regenerative medicine. Traditional tissue culture uses fetal bovine serum (FBS), which complicates utilization of ASCs in human medicine. Human platelet lysate (hPL) is one potential xeno-free, alternative supplement for use in ASC culture. In this study, adipogenic and osteogenic differentiation in media supplemented with 10% FBS or 10% hPL was compared in human ASCs derived from abdominoplasty (HAP) or from adipose associated with debrided burned skin (BH). Most (95-99%) cells cultured in FBS were stained positive for CD73, CD90, CD105, and CD142. FBS supplementation was associated with increased triglyceride content and expression of adipogenic genes. Culture in hPL significantly decreased surface staining of CD105 by 31% and 48% and CD142 by 27% and 35% in HAP and BH, respectively (p < 0.05). Culture of BH-ASCs in hPL also increased expression of markers of osteogenesis and increased ALP activity. These data indicate that application of ASCs for wound healing may be influenced by ASC source as well as culture conditions used to expand them. As such, these factors must be taken into consideration before ASCs are used for regenerative purposes.
Project description:Immune cell therapy has emerged as a promising approach to treat malignancies that were up until recently only treated on a palliative basis. Chimeric antigen receptor- (CAR-) modified T lymphocytes (T cells) in particular have proven to be very effective for certain hematological malignancies. The production of CAR T cells usually involves viral transduction and <i>ex vivo</i> culture of T cells. The aim of this study was to explore the use of human platelet lysate (HPL) compared to two commonly used supplements, human AB serum (ABS) and fetal bovine serum (FBS), for modified T cell production. For studying transduction, activated T cells were transduced with lentivirus to deliver GFP transgenes with three different promoters. Transduction efficiency (percent GFP) was similar among the supplements, and a modest increase in the transgene product (mean fluorescence intensity) was observed when HPL was used as a supplement compared to ABS. To study the effect of supplements on expansion, peripheral blood mononuclear cells (PBMCs) were activated and expanded in the presence of interleukin 2 (IL2) for fourteen days. T cell expansions using HPL and ABS were comparable and slightly less than the expansion obtained with FBS. Interestingly, cells expanded in media supplemented with HPL showed a higher percentage of T cells with a central memory phenotype compared to those expanded in ABS or FBS. Protein profiling revealed that the phenotypic differences may be explained by elevated levels of several cytokines in HPL, including IL7. The results suggest that the use of HPL as a cell culture supplement during the production of modified T cells is a reasonable alternative to ABS. Furthermore, the use of HPL may enhance <i>in vivo</i> performance of the final product by enriching for central memory T cells that are associated with long-term persistence following adoptive transfer.
Project description:Pooled human platelet lysate (pHPL) is increasingly used as replacement of animal serum for manufacturing of stromal cell therapeutics. Porcine heparin is commonly applied to avoid clotting of pHPL-supplemented medium but the influence of heparin on cell behavior is still unclear. Aim of this study was to investigate cellular uptake of heparin by fluoresceinamine-labeling and its impact on expression of genes, proteins and function of human stromal cells derived from bone marrow (BM), umbilical cord (UC) and white adipose tissue (WAT). Cells were isolated and propagated using various pHPL-supplemented media with or without heparin. Flow cytometry and immunocytochemistry showed differential cellular internalization and lysosomal accumulation of heparin. Transcriptome profiling revealed regulation of distinct gene sets by heparin including signaling cascades involved in proliferation, cell adhesion, apoptosis, inflammation and angiogenesis, depending on stromal cell origin. The influence of heparin on the WNT, PDGF, NOTCH and TGFbeta signaling pathways was further analyzed by a bead-based western blot revealing most alterations in BM-derived stromal cells. Despite these observations heparin had no substantial effect on long-term proliferation and in vitro tri-lineage differentiation of stromal cells, indicating compatibility for clinically applied cell products.
Project description:Adipose-derived mesenchymal stem cells (ADMSCs) are a promising strategy for orthopaedic applications, particularly in bone repair. Ex vivo expansion of ADMSCs is required to obtain sufficient cell numbers. Xenogenic supplements should be avoided in order to minimise the risk of infections and immunological reactions. Human platelet lysate and human plasma may be an excellent material source for ADMSC expansion. In the present study, use of blood products after their recommended transfusion date to prepare human platelet lysate (HPL) and human plasma (Hplasma) was evaluated for in vitro culture expansion and osteogenesis of ADMSCs.Human ADMSCs were cultured in medium supplemented with HPL, Hplasma and a combination of HPL and Hplasma (HPL+Hplasma). Characteristics of these ADMSCs, including osteogenesis, were evaluated in comparison with those cultured in fetal bovine serum (FBS).HPL and HPL+Hplasma had a significantly greater growth-promoting effect than FBS, while Hplasma exhibited a similar growth-promoting effect to that of FBS. ADMSCs cultured in HPL and/or Hplasma generated more colony-forming unit fibroblasts (CFU-F) than those cultured in FBS. After long-term culture, ADMSCs cultured in HPL and/or Hplasma showed reduced cellular senescence, retained typical cell phenotypes, and retained differentiation capacities into osteogenic and adipogenic lineages.HPL and Hplasma prepared from blood products after their recommended transfusion date can be used as an alternative and effective source for large-scale ex vivo expansion of ADMSCs.Cite this article: J. Phetfong, T. Tawonsawatruk, K. Seenprachawong, A. Srisarin, C. Isarankura-Na-Ayudhya, A. Supokawej. Re-using blood products as an alternative supplement in the optimisation of clinical-grade adipose-derived mesenchymal stem cell culture. Bone Joint Res 2017;6:414-422. DOI: 10.1302/2046-3758.67.BJR-2016-0342.R1.
Project description:BACKGROUND:The autologous nerve graft, despite its donor site morbidity and unpredictable functional recovery, continues to be the gold standard in peripheral nerve repair. Rodent research studies have shown promising results with cell transplantation of human adipose-derived stem cells (hADSC) in a bioengineered conduit, as an alternative strategy for nerve regeneration. To achieve meaningful clinical translation, cell therapy must comply with biosafety. Cell extraction and expansion methods that use animal-derived products, including enzymatic adipose tissue dissociation and the use of fetal bovine serum (FBS) as a culture medium supplement, have the potential for transmission of zoonotic infectious and immunogenicity. Human-platelet-lysate (hPL) serum has been used in recent years in human cell expansion, showing reliability in clinical applications. METHODS:We investigated whether hADSC can be routinely isolated and cultured in a completely xenogeneic-free way (using hPL culture medium supplement and avoiding collagenase digestion) without altering their physiology and stem properties. Outcomes in terms of stem marker expression (CD105, CD90, CD73) and the osteocyte/adipocyte differentiation capacity were compared with classical collagenase digestion and FBS-supplemented hADSC expansion. RESULTS:We found no significant differences between the two examined extraction and culture protocols in terms of cluster differentiation (CD) marker expression and stem cell plasticity, while hADSC in hPL showed a significantly higher proliferation rate when compared with the usual FBS-added medium. Considering the important key growth factors (particularly brain-derived growth factor (BDNF)) present in hPL, we investigated a possible neurogenic commitment of hADSC when cultured with hPL. Interestingly, hADSC cultured in hPL showed a statistically higher secretion of neurotrophic factors BDNF, glial cell-derived growth factor (GDNF), and nerve-derived growth factor (NFG) than FBS-cultured cells. When cocultured in the presence of primary neurons, hADSC which had been grown under hPL supplementation, showed significantly enhanced neurotrophic properties. CONCLUSIONS:The hPL-supplement medium could improve cell proliferation and neurotropism while maintaining stable cell properties, showing effectiveness in clinical translation and significant potential in peripheral nerve research.
Project description:Gingiva has been identified as a minimally invasive source of multipotent progenitor cells (GPCs) for use in bone tissue engineering (BTE). To facilitate clinical translation, it is important to characterize GPCs in xeno-free cultures. Recent evidence indicates several advantages of three-dimensional (3D) spheroid cultures of mesenchymal stromal cells (MSCs) over conventional 2D monolayers. The present study aimed to characterize human GPCs in xeno-free 2D cultures, and to test their osteogenic potential in 3D cultures, in comparison to bone marrow MSCs (BMSCs). Primary GPCs and BMSCs were expanded in human platelet lysate (HPL) or fetal bovine serum (FBS) and characterized based on in vitro proliferation, immunophenotype and multi-lineage differentiation. Next, 3D spheroids of GPCs and BMSCs were formed via self-assembly and cultured in HPL. Expression of stemness- (SOX2, OCT4, NANOG) and osteogenesis-related markers (BMP2, RUNX2, OPN, OCN) was assessed at gene and protein levels in 3D and 2D cultures. The cytokine profile of 3D and 2D GPCs and BMSCs was assessed via a multiplex immunoassay. Monolayer GPCs in both HPL and FBS demonstrated a characteristic MSC-like immunophenotype and multi-lineage differentiation; osteogenic differentiation of GPCs was enhanced in HPL vs. FBS. CD271+ GPCs in HPL spontaneously acquired a neuronal phenotype and strongly expressed neuronal/glial markers. 3D spheroids of GPCs and BMSCs with high cell viability were formed in HPL media. Expression of stemness- and osteogenesis-related genes was significantly upregulated in 3D vs. 2D GPCs/BMSCs; the latter was independent of osteogenic induction. Synthesis of SOX2, BMP2 and OCN was confirmed via immunostaining, and in vitro mineralization via Alizarin red staining. Finally, secretion of several growth factors and chemokines was enhanced in GPC/BMSC spheroids, while that of pro-inflammatory cytokines was reduced, compared to monolayers. In summary, monolayer GPCs expanded in HPL demonstrate enhanced osteogenic differentiation potential, comparable to that of BMSCs. Xeno-free spheroid culture further enhances stemness- and osteogenesis-related gene expression, and cytokine secretion in GPCs, comparable to that of BMSCs.