Maintenance of hPSCs under Xeno-Free and Chemically Defined Culture Conditions.
Ontology highlight
ABSTRACT:
SUBMITTER: Lim JJ
PROVIDER: S-EPMC6881038 | biostudies-literature |
REPOSITORIES: biostudies-literature
Similar Datasets
Project description:Extended pluripotent stem (EPS) cells have shown great applicative potentials in generating synthetic embryos, directed differentiation and disease modeling. However, the lack of a xeno-free culture condition has significantly limited their applications. Here, we report a chemically defined and xeno-free culture system for culturing and deriving human EPS cells in vitro. Xeno-free human EPS cells can be long-term and genetically stably maintained in vitro, as well as preserve their embryonic and extraembryonic developmental potentials. Furthermore, the xeno-free culturing system also permits efficient derivation of human EPS cells from human fibroblast through reprogramming. Our study could have broad utility in future applications of human EPS cells in biomedicine.
| S-EPMC8139978 | biostudies-literature
Project description:Adipose tissue is an abundant source of stem cells. However, liposuction cannot yield cell quantities sufficient for direct applications in regenerative medicine. Therefore, the development of GMP-compliant ex vivo expansion protocols is required to ensure the production of a "cell drug" that is safe, reproducible, and cost-effective. Thus, we developed our own basal defined xeno- and serum-free cell culture medium (UrSuppe), specifically formulated to grow human adipose stem cells (hASCs). With this medium, we can directly culture the stromal vascular fraction (SVF) cells in defined cell culture conditions to obtain hASCs. Cells proliferate while remaining undifferentiated, as shown by Flow Cytometry (FACS), Quantitative Reverse Transcription PCR (RT-qPCR) assays, and their secretion products. Using the UrSuppe cell culture medium, maximum cell densities between 0.51 and 0.80 × 105 cells/cm2 (=2.55-4.00 × 105 cells/mL) were obtained. As the expansion of hASCs represents only the first step in a cell therapeutic protocol or further basic research studies, we formulated two chemically defined media to differentiate the expanded hASCs in white or beige/brown adipocytes. These new media could help translate research projects into the clinical application of hASCs and study ex vivo the biology in healthy and dysfunctional states of adipocytes and their precursors. Following the cell culture system developers' practice and obvious reasons related to the formulas' patentability, the defined media's composition will not be disclosed in this study.
| S-EPMC7926673 | biostudies-literature
Project description:Non-human primates (NHPs) are pivotal animal models for translating novel cell replacement therapies into clinical applications, including validating the safety and efficacy of induced pluripotent stem cell (iPSC)-derived products. Preclinical development and the testing of cell-based therapies ideally comprise xenogeneic (human stem cells into NHPs) and allogenic (NHP stem cells into NHPs) transplantation studies. For the allogeneic approach, it is necessary to generate NHP-iPSCs with generally equivalent quality to the human counterparts that will be used later on in patients. Here, we report the generation and characterization of transgene- and feeder-free cynomolgus monkey (Macaca fascicularis) iPSCs (Cyno-iPSCs). These novel cell lines have been generated according to a previously developed protocol for the generation of rhesus macaque, baboon, and human iPSC lines. Beyond their generation, we demonstrate the potential of the novel Cyno-iPSCs to differentiate into two clinically relevant cell types, i.e., cardiomyocytes and neurons. Overall, we provide a resource of novel iPSCs from the most frequently used NHP species in the regulatory testing of biologics and classical pharmaceutics to expand our panel of iPSC lines from NHP species with high relevance in preclinical testing and translational research.
| S-EPMC10969578 | biostudies-literature
Project description:Defects in neural crest development have been implicated in many human disorders, but information about human neural crest formation mostly depends on extrapolation from model organisms. Human pluripotent stem cells (hPSCs) can be differentiated into in vitro counterparts of the neural crest, and some of the signals known to induce neural crest formation in vivo are required during this process. However, the protocols in current use tend to produce variable results, and there is no consensus as to the precise signals required for optimal neural crest differentiation. Using a fully defined culture system, we have now found that the efficient differentiation of hPSCs to neural crest depends on precise levels of BMP signaling, which are vulnerable to fluctuations in endogenous BMP production. We present a method that controls for this phenomenon and could be applied to other systems where endogenous signaling can also affect the outcome of differentiation protocols.
| S-EPMC5639211 | biostudies-literature
Project description:Outgrowths of limbal epithelium by explant culture are used to treat limbal stem cell deficiency (LSCD). The explant culture medium is always complemented with serum, a complex solution which includes TGF?. Since TGF? is a cytostatic effector for epithelial proliferation we examined its effect on these cultures. Limbal biopsies were set on explant culture in DMEM/F12 with 5 ng/ml EGF and cholera toxin (ChT), ITS, and 5% FBS, henceforth SHEM or a) SHEMSB=SHEM plus SB431542 an inhibitor of TGF? signaling; b) sfSHEM = SHEM with FBS replaced by 0.05% Albumax II; and c) sfSHEMSB and sfSHEMA83 = sfSHEM plus, respectively, SB431542 or A-83-01, another TGF? inhibitor. After the initial outgrowths reached 3 cm in diameter, the limbal biopsies were serially transferred up to six times onto new inserts. Biopsy explant outgrowths were trypsinized and cell yield, morphology and stem-cell related JC-1 exclusion (IOVS, 52:4330) were determined by flow cytometry. Cells we plated at low density seeding to compare relative clonal proliferative activity. The expression of three proteins whose levels are associated with growth and differentiation states, Krt3, connexin 43 and p63 were determined by immunohistology and/or Western blot. Cell yield in rabbit, relative to SHEM (in %) were, SHEMSB, 104 ± 13 (p > 0.95); sfSHEM: 5 ± 3; and sfSHEMSB, 94 ± 18 (p > 0.95). Cell size and morphology, JC1 dye exclusion, Krt3, p63 and connexin 43 content, proliferation efficiency and the preservation of extended proliferative potential of the serially cultured biopsies were similar for SHEM, SHEMSB and sfSHEMSB. The only differences observed where reduced expression of Krt3 and increased preservation of p63 in the FBS-free medium. Removal of EGF from sfSHEMSB reduced yield by 92 ± 6% (p < 0.05). Removal of Albumax and ChT to establish a xeno-free medium caused a small, non-statistical decrease in growth rates. Equivalent results were observed in a preliminary experiment in human. These results suggest that in the absence serum endogenously generated TGF? act as an autocrine cytostatic agent and that TGF? inhibitors allow explant culture in xeno-free, chemically defined medium. Furthermore, the pro-growth effect of serum in limbal explant cultures may result exclusively from neutralization of the TGF? cytostatic effect.
| S-EPMC4842090 | biostudies-literature
Project description:Large-scale production of human induced pluripotent stem cells (hiPSCs) by robust and economic methods has been one of the major challenges for translational realization of hiPSC technology. Here we demonstrate a scalable culture system for hiPSC expansion using the E8 chemically defined and xeno-free medium under either adherent or suspension conditions. To optimize suspension conditions guided by a computational simulation, we developed a method to efficiently expand hiPSCs as undifferentiated aggregates in spinner flasks. Serial passaging of two different hiPSC lines in the spinner flasks using the E8 medium preserved their normal karyotype and expression of undifferentiated state markers of TRA-1-60, SSEA4, OCT4, and NANOG. The hiPSCs cultured in spinner flasks for more than 10 passages not only could be remained pluripotent as indicated by in vitro and in vivo assays, but also could be efficiently induced toward mesodermal and hematopoietic differentiation. Furthermore, we established a xeno-free protocol of single-cell cryopreservation and recovery for the scalable production of hiPSCs in spinner flasks. This system is the first to enable an efficient scale-up bioprocess in completely xeno-free condition for the expansion and cryopreservation of hiPSCs with the quantity and quality compliant for clinical applications.
| S-EPMC4628790 | biostudies-literature
Project description:Cell therapy is witnessing a notable shift toward cell-free treatments based on paracrine factors, in particular, towards small extracellular vesicles (sEV), that mimic the functional effect of the parental cells. While numerous sEV-based applications are currently in advanced preclinical stages, their promised translation depends on overcoming the manufacturing hurdles posed by the large-scale production of purified sEV. Unquestionably, the culture medium used with the parental cells plays a key role in the sEV's secretion rate and content. An essential requisite is the use of a serum-, xeno-, and blood-free medium to meet the regulatory entity requirements of clinical-grade sEV's production. Here, we evaluated OxiumTMEXO, a regulatory complying medium, with respect to production capacity and conservation of the EV's characteristics and functionality and the parental cell's phenotype and viability. A comparative study was established with standard DMEM and a commercially available culture medium developed specifically for sEV production. Under similar conditions, OxiumTMEXO displayed a three-fold increase of sEV secretion, with an enrichment of particles ranging between 51 and 200 nm. These results were obtained through direct quantification from the conditioned medium to avoid the isolation method's interference and variability and were compared to the two culture media under evaluation. The higher yield obtained was consistent with several harvest time points (2, 4, and 6 days) and different cell sources, incluiding umbilical cord-, menstrual blood-derived mesenchymal stromal cells and fibroblasts. Additionally, the stem cell phenotype and viability of the parental cell remained unchanged. Furthermore, OxiumTMEXO-sEV showed a similar expression pattern of the vesicular markers CD63, CD9, and CD81, with respect to sEV derived from the other conditions. The in vitro internalization assays in different target cell types and the pharmacokinetic profile of intraperitoneally administered sEV in vivo indicated that the higher EV production rate did not affect the uptake kinetics or the systemic biodistribution in healthy mice. In conclusion, the OxiumTMEXO medium sustains an efficient and robust production of large quantities of sEV, conserving the classic functional properties of internalization into acceptor target cells and biodistribution in vivo, supplying the amount and quality of EVs for the development of cell-free therapies.
| S-EPMC8187876 | biostudies-literature
Project description:ObjectivesUmbilical cord mesenchymal stromal cells (UCMSCs) can be considered to become a new gold standard for MSC-based therapies. A serum and xeno-free, chemically defined and no-plate-coating-based culture system will greatly facilitate development of robust, clinically acceptable bioprocesses for reproducibly generating quality-assured UCMSCs.Materials and methodsIn this study, we report for the first time, such a serum-free, xeno-free, completely chemically defined and no-plate-coating-based culture system for the isolation and expansion of UCMSCs, whose biological characteristics were evaluated and compared with serum-containing medium (SCM) methods.ResultsThis culture system not only supported UCMSC primary cultures but also allowed for their expansion at low seeding density. Compared to SCM, UCMSCs in SFM exhibited (i) higher proliferative and colony-forming capacities; (ii) distinctly different morphologies; (iii) similar phenotype; (iv) similar pluripotency-associated marker expression; (v) superior osteogenic, but reduced adipogenic differentiation capacitities. In addition, UCMSCs cultured in SFM retained similar immunomodulatory properties to those in SCM.ConclusionsOur findings demonstrate the feasibility of isolating and expanding UCMSCs in a completely serum-free, xeno-free, chemically defined and no-plate-coating-based culture system and represent an important step forward for development of robust, clinically acceptable bioprocesses for UCMSCs. Further, this provides a superior study platform for UCMSCs biology in a controlled environment.
| S-EPMC6496339 | biostudies-literature
Project description:We re-examine the individual components for human embryonic stem cell (ESC) and induced pluripotent stem cell (iPSC) culture and formulate a cell culture system in which all protein reagents for liquid media, attachment surfaces and splitting are chemically defined. A major improvement is the lack of a serum albumin component, as variations in either animal- or human-sourced albumin batches have previously plagued human ESC and iPSC culture with inconsistencies. Using this new medium (E8) and vitronectin-coated surfaces, we demonstrate improved derivation efficiencies of vector-free human iPSCs with an episomal approach. This simplified E8 medium should facilitate both the research use and clinical applications of human ESCs and iPSCs and their derivatives, and should be applicable to other reprogramming methods.
| S-EPMC3084903 | biostudies-literature
Project description:Human trophoblast stem cells (hTSCs) have emerged as a powerful tool to model early placental development in vitro. Analogous to the epithelial cytotrophoblast in the placenta, hTSCs can differentiate into cells of the extravillous trophoblast (EVT) lineage or the multinucleate syncytiotrophoblast (STB). Here we present a chemically defined culture system for STB and EVT differentiation of hTSCs. Notably, in contrast to current approaches, we neither utilize forskolin for STB formation nor transforming growth factor-beta (TGFβ) inhibitors or a passage step for EVT differentiation. Strikingly, the presence of a single additional extracellular cue-laminin-111-switched the terminal differentiation of hTSCs from STB to the EVT lineage under these conditions. In the absence of laminin-111, STB formation occurred, with cell fusion comparable to that obtained with differentiation mediated by forskolin; however, in the presence of laminin-111, hTSCs differentiated to the EVT lineage. Protein expression of nuclear hypoxia-inducible factors (HIF1α and HIF2α) was upregulated during EVT differentiation mediated by laminin-111 exposure. A heterogeneous mixture of Notch1+ EVTs in colonies and HLA-G+ single-cell EVTs were obtained without a passage step, reminiscent of heterogeneity in vivo. Further analysis showed that inhibition of TGFβ signaling affected both STB and EVT differentiation mediated by laminin-111 exposure. TGFβ inhibition during EVT differentiation resulted in decreased HLA-G expression and increased Notch1 expression. On the other hand, TGFβ inhibition prevented STB formation. The chemically defined culture system for hTSC differentiation established herein facilitates quantitative analysis of heterogeneity that arises during hTSC differentiation and will enable mechanistic studies in vitro.
| S-EPMC10176266 | biostudies-literature