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Karyotypic analysis of human pluripotent stem cell lines cocultured with inactivated HFF-hUCS. The karyotypic stability of hPSCs after prolonged coculture with inactivated HFF-hUCS was detected using the G-banding method. hPSC lines cocultured with inactivated HFF-hUCS maintained a normal karyotype similar to hPSCs lines cocultured with inactivated HFF-FBS. Chula2.hES maintained the 46,XY karyotype and HFSK#11.hiPS maintained the 46,XY karyotype on both types of feeder cells. iHFF-hUCS = inactivated HFF-hUCS and iHFF-FBS = inactivated HFF-FBS.
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Although human pluripotent stem cells (hPSCs) can proliferate robustly on the feeder-free culture system, genetic instability of hPSCs has been reported in such environment. Alternatively, feeder cells enable hPSCs to maintain their pluripotency. The feeder cells are usually grown in a culture medium containing fetal bovine serum (FBS) prior to coc...
Similar publications
One model to study the emergence of the human trophoblast (TB) has been the exposure of pluripotent stem cells to bone morphogenetic protein 4 (BMP4) in presence of inhibitors of ACTIVIN/TGFB; A83-01 and FGF2; PD173074 (BAP), which generates a mixture of cytotrophoblast, syncytiotrophoblast, and cells with similarities to extravillous trophoblast....
Citations
... Ebrahim et al. [104] reported that even the human umbilical cord MSCs derived EVs along with estrogen treatment can ameliorate IUA symptoms in rats. Even transplanting extracellular vesicles or exosomes derived from MSCs suffice to improve and rejuvenate the endometrium in animal models and in clinical studies [105][106][107][108]. This clearly suggests that beneficial effects of MSCs are exclusively due to their ability to provide paracrine support. ...
Compared to embryonic and induced pluripotent stem cells, mesenchymal stem/stromal cells (MSCs) have made their presence felt with good therapeutic promise and safety profile. Transplanting MSCs has successfully helped to reverse infertility and resulted in live births in animal models and also in humans. But the underlying mechanism for their therapeutic potential is not yet clear. MSCs are not pluripotent and hence lack plasticity to differentiate into multiple adult cell types. They rather act as ‘paracrine providers’ to the tissue-resident stem cells since similar beneficial effects are also observed when their secretome (microvesicles or exosomes) is transplanted. Cytokines, growth factors, signaling lipids, mRNAs, and miRNAs secreted by MSCs enables tissue-resident stem cells to undergo differentiation into specific cell types. Tissue-resident stem cells include pluripotent, very small embryonic-like stem cells (VSELs) and progenitors [spermatogonial (SSCs), ovarian (OSCs) and endometrial (EnSCs) stem cells in testes, ovary and uterus respectively] which function in a subtle manner to maintain life-long tissue homeostasis and regenerate damaged (non-functional) reproductive tissues by differentiating into sperm, oocytes and endometrial epithelial cells respectively. Similar to restoring spermatogenesis, primordial follicles numbers are increased upon transplanting MSCs. Published literature suggests that MSCs do not differentiate into epithelial cells in the endometrium. Nuclear OCT-4 positive VSELs and cytoplasmic OCT-4, AXIN2 and KERATIN-19 positive epithelial progenitors have a greater role during endometrial regeneration. We propose, transplantation of MSCs simply provides growth factors/cytokines essential for the tissue-resident stem/progenitor cells to undergo differentiation into sperm, eggs and endometrial epithelial cells in the reproductive tissues.
... In recent years, cord blood serum is mainly preferred as a reasonable supplement for different sources-derived mesenchymal stem cells (bone-marrow mesenchymal stem cells, placenta-derived mesenchymal stem cells, Wharton's jelly mesenchymal stem cells and cord blood mesenchymal stem cells) proliferation, osteogenic and adipogenic differentiation and migration (13,28,(33)(34)(35). Moreover, in fibroblast cell cultures, human cord serum potentially induces higher proliferation rate than FBS (36). For ocular surface epithelial cells (limbal, conjunctival, and cornea epithelial cells) proliferation and differentiation, cord serum is established as an effective and safer alternative growth media supplement in culture systems (37,38). ...
Regenerative medicine is a branch of medicine that incorporates tissue-engineering, biomaterials, and cell therapy approaches to replace or repair damaged cells and tissues. Umbilical cord serum (UCS) is an important liquid component of cord blood, which has a reliable source of innumerable growth factors and biologically active molecules. Usually, serum can be prepared from different sources of blood. In therapeutic application, cord serum can be prepared and used in the form of eye drops for the treatment of severe dry eye diseases, ocular burns, glaucoma, persistent corneal epithelial defects and neurotrophic keratitis. In addition, cord serum combined with synthetic bio scaffold materials is used to regenerate different types of tissues including tympanic membrane regeneration, bone regeneration and nerve regeneration. Absence of animal origin viruses and bacteria, lack of xenoproteins and cost-effective features make cord serum a feasible choice as replacement of fetal bovine serum in cell culture techniques. Thus, this review emphasizes the role of cord serum in regenerative therapy and clinical uses.
... Cytogenetic analysis was performed on MSC at passage 25 to evaluate chromosome stability by conventional karyotype and the standard procedure followed [15]. ...
Background
Recent studies have revealed that the therapeutic effect of mesenchymal stem cells (MSCs) is due to their secretome. Secretome is considered to be advantageous over cells, because of less chances of teratoma formation and no cell genome variability of donor and recipient. The current study aimed to screen the secretome of cord lining (CL) and Wharton jelly (WJ) of human umbilical cord stem cells and their functionality.
Methods
Explants culture of human CL and WJ were characterized for classical MSC markers, pluripotency for germ layer expression then studied for their neurosphere and neurogenesis ability. These stem cells were screened for the expression of trophic factors (BDNF/GDNF/CNTF/NT3/NT4/NGF/FGF1/FGF2/VEGF/HGF/IGF/EGF/PlGF/IFN gamma/TNF-α/IL-1b/IL-4/IL-10/IFN-α1/TGF-β). Cells were cultured under serum-free conditions for 48 h and the conditioned media (CM) were collected. Functional effect of CM was analyzed on SHSY5Y, U87MG, and endothelial cells for proliferation/differentiation/anti-inflammatory and pro-angiogenic effects respectively.
Results
CL and WJ cells showed positive for MSC markers and negative for hematopoietic lineage. Both stem cells exhibited highly proliferative phenotype with normal karyotype even after several passages. All the major growth factors were expressed at varying levels expect CNTF. Cells illustrated the expression of various inflammatory modulators except IL-10 and elucidated anti-inflammatory and pro-angiogenic effect. These cells formed neurosphere in vitro and upon neuronal induction, expressed mature neuronal markers. CM enhanced SHSY5Y cell proliferation and differentiation.
Conclusion
Easily accessible human umbilical cord stem’s secretome can have potential therapeutic effect in neurodegenerative disease.
Lay Summary
Cellular secretions from human umbilical cord stem cells have the potential property to exert neuroprotective effect. Hence, instead of using cells as the therapeutics, the cellular secretions can be potentially used to achieve clinical healing/repair of degenerative tissues of our nervous system.
... To address all these risks, regulatory authorities, industry and the research community demand suitable alternatives to provide safe, regulated and effective cell therapy products to patients [18]. Currently, many autologous or allogenic human blood-derived products such as human serum [19,20], platelet-rich plasma (PRP) [21], platelet lysate [14,[22][23][24] or umbilical cord blood serum [25,26] are being explored as potential FBS replacement. Regarding PRPs, diverse protocols are being performed leading to multiple commercially available products, which methodological and composition variability contribute to the heterogeneity of the final therapeutic outcomes. ...
Aim:
This study investigated the use of the autologous technology of plasma rich in growth factors (PRGF) as a human-based substitute to fetal bovine serum (FBS) in the culture of human dental pulp stem cells.
Materials & methods:
Stem cell characterization was performed. Analysis of isolation, proliferation, migration, trilineage differentiation, senescence and cryopreservation were compared between FBS and PRGF.
Results:
Human dental pulp stem cell cultures isolated and maintained with PRGF showed a significantly higher number of cells per explant than FBS cultures. Cell proliferation, migration, osteogenic mineralization and adipogenic differentiation were found to be significantly higher in PRGF than FBS.
Conclusion:
The autologous PRGF technology could be a suitable and safer substitute for FBS as a culture medium supplement for clinical translation of cell therapy.
... In addition, the hUCS-HFF medium maintains the pluripotency and karyotypic stability of hESCs over a long period. 47 Thus, it is necessary to search for a better standardization and choice for free alternatives of fetal bovine serum, and HFF are presented as an alternative to avoid the application of substances and cells of animal origin in the culture depend- ent on hESC feeder medium. ...
Circumcision is one of the most performed surgical procedures worldwide, and it is estimated that one in three men worldwide is circumcised, which makes the preputial skin removed after surgery an abundant material for possible applications. In particular, it is possible efficiently to isolate the cells of the foreskin, with fibroblasts being the most abundant cells of the dermis and the most used in biomedical research. This work aimed to review the knowledge and obtain a broad view of the main applications of human foreskin fibroblast cell culture. A literature search was conducted, including clinical trials, preclinical basic research studies, reviews and experimental studies. Several medical and laboratory applications of human foreskin fibroblast cell culture have been described, especially when it comes to the use of human foreskin fibroblasts as feeder cells for the cultivation of human embryonic stem cells, in addition to co-culture with other cell types. The culture of foreskin fibroblasts has also been used to: obtain induced pluripotent stem cells; the diagnosis of Clostridium difficile; to test the toxicity and effect of substances on normal cells, especially the toxicity of possible antineoplastic drugs; in viral culture, mainly of the human cytomegalovirus, study of the pathogenesis of other microorganisms; varied studies of cellular physiology and cellular interactions. Fibroblasts are important for cell models for varied application cultures, demonstrating how the preputial material can be reused, making possible new applications.
Level of evidence: Not applicable for this multicentre audit.
... Not only for cell growth, human pluripotent stem cell lines could maintain their pluripotencies, differentiation capacities, and karyotypic stabilities after being co-cultured for extended period with hUCBS. 24,27 In addition, hUCBS cryopreservation has been performed extensively with modified methods. 24,28 Nevertheless, hUCBS is also useful when cells were induced for differentiation. ...
Background: Cytokines and growth factors were reported to play an important role in stimulating fibroblast proliferation. In vitro culture, fibroblast is mostly culture in medium containing fetal bovine serum (FBS). Human umbilical cord blood (hUCB) has been reported to have low immunogenic property and potential in wound healing, so therefore hUCB serum (hUCBS) could be potential and were investigated in current study.
Materials and Methods: Five hUCBs were collected from healthy volunteers with normal delivering procedure. hUCB was ex utero immediately collected from umbilical vein in vacutainers and processed. NIH3T3 cells were cultured in DMEM with 10% FBS or 5-20% hUCBS for 48 hours. Cells were then quantified using MTT assay. Protein concentration of FBS and hUCBS were quantified using Bradford assay.
Results: NIH3T3 cells density grown in DMEM with 10% FBS was the lowest. NIH3T3 cells densities were increased along with the increment of hUCBS concentrations. MTT results showed that average number of NIH3T3 cells grown in DMEM with 10% FBS was 6,185±1,243. Meanwhile average numbers of NIH3T3 cells grown in DMEM with 5%, 10% and 20% hUCBS were 8,126±628, 9,685±313 and 12,200±304, respectively. Average numbers of NIH3T3 cells grown in DMEM with 5% hUCBS were significantly higher than the ones with 10% FBS (p=0.000). Bradford results showed that concentration of hUCBS was significantly higher than the one of FBS (p=0.000).
Conclusion: hUCBS could induce higher proliferation rate of NIH3T3 cells than FBS. Hence hUCBS could be suggested as an alternate of FBS in inducing fibroblast.
Keywords: NIH3T3, fibroblast, UCB, serum, FBS, proliferation
Although BHPF has been widely used in plastic manufacturing as a substitute for BPA, current evidence suggests that BHPF also causes harmful effects on reproduction. However, effects of BHPF on mammalian early pregnancy are still poorly defined. This study aimed to explore the effects of BHPF on early pregnancy, especially decidualization and embryonic development in mice and human beings. The results showed that 50 and 100 mg/kg BHPF exposure reduced birth weight, and implantation site weight on the day 8 of pregnancy in mice. Because BHPF inhibits both embryo development and artificial decidualization in mice, suggesting that the detrimental effects of BHPF should be from its effects on embryo development and decidualization. Under in vitro decidualization, 10 μM BHPF inhibits decidualization and leads to disordered expression of Lamin B1 and collagen in mice. In addition, 10 μM BHPF also inhibits decidualization, and causes disordered expression of both collagen III and Lamin B1 under human in vitro decidualization. However, collagen III supplement can rescue BHPF inhibition on decidualization. Further, our study demonstrated that BHPF impairs human decidualization through the HB-EGF/EGFR/STAT3/Collagen III pathway. Taken together these data suggest that exposure to BHPF impairs decidualization during early pregnancy.
Background
In recent years, there has been a significant decline in the demand for cord blood units (CBUs). This trend has led to cord blood banks (CBBs) exploring complementary uses of CBUs in order to exploit the full potential of this unique, valuable, and readily available product. The aim of this study was to establish standardized protocols for the preparation of four cord blood (CB) derivatives: plasma (CB-P), serum (CB-S), induced-serum (CB-IS) and growth factor-rich plasma (GFRP); to measure their respective growth-factor content and their potential as cell culture supplements, and finally, to identify the characteristics of each derivative beyond their growth-factor composition, in the context of optimizing CBBs resources.
Study design and method
To this end, CB plasma and serum were prepared and their concentrations for ten growth factors (TGF-β1, TGF-β2, TGF-β3, EGF, HGF, PDGF-BB, VEGF-A, VEGF-D, IGF-I, IGF-II) were determined using a multiplex bead array, and compared to adult plasma and serum. Protocols for the preparation of CB-IS and GFRP with reverse anticoagulation using CaCl2·2H2O were also established, and all four derivatives were compared for their EGF and TGF-β1 content by enzyme-linked immunosorbent assay (ELISA), and also for their cell culture supplement capacity.
Results
CB plasma was shown to be richer than adult plasma for TGF-β2 and TGF-β3, with a lower concentration of IGF-I and IGF-II. CB serum was shown to be richer than adult serum for TGF-β2, TGF-β3, EGF, HGF, PDGF-BB, and VEGF-A and D, and poorer in IGF-I and II. The measure of EGF and TGF-β1 concentrations has shown that CB serum is the most concentrated (1874 pg/ml and 41 094 pg/ml) of the CB derivatives, followed by induced-serum (405 pg/ml and 16 735 pg/ml), growth factor-rich plasma (131 and 7947 pg/ml) and plasma (8 and 2845 pg/ml). All four CB derivatives were shown to be superior to FBS in sustaining cell growth at low doses.
Conclusion
Our study shows that four CB derivatives can be easily prepared and pooled to provide significant volume of products that vary in their growth factor composition. A cord blood bank interested in introducing such manufacturing will need to evaluate the financial and processing characteristics of each derivative. The use of standardized manufacturing protocols such as the ones we suggest could help research initiatives exploring the potential therapeutic uses of such rich and high-quality starting material.
Human ovarian follicular fluid (HOFF) contains proteins, extracellular matrixes necessary for growth and maturation of oocytes as well as granulosa cells. Epithelial cells and stem cells can be isolated from HOFF. However, information regarding stem cells derived from HOFF is still lacking. The objectives of the present study were to isolate, characterize, and differentiate cells derived from HOFF. HOFF was collected during the routine aspiration of oocytes in an assisted fertilization program and subjected to cell isolation, characterization, and in vitro culture. After 24 h of culture, different cell morphologies including epithelial-like-, neural-like- and fibroblast-like cells were observed. Immunocytochemistry reveals the expression of pluripotent stem cell markers (OCT4, NANOG, SSEA4), epithelial marker (CK18), FSH- and LH-receptor. For in vitro culture, the isolated cells were continuously cultured in a growth medium; alpha MEM containing 10% FBS and epidermal growth factor (EGF). After 2 weeks of in vitro culture, cells with fibroblast-like morphology dominantly grow in the culture vessels and resemble mesenchymal stem cells (MSCs). HOFF-derived cells exhibited MSC expression of CD44, CD73, CD90, CD105, CD146, and STRO-1, and were capable of differentiation into osteoblasts, chondrocytes, and adipocytes. After induction of neural differentiation, HOFF-derived cells formed spheroidal structures and expressed neural stem cell markers including Nestin, β-tubulin III, and O4. Besides, the oocyte-like structure was observed after prolonged culture of HOFF. In conclusion, cells derived from follicular fluid exhibited stem cell characteristics, which could be useful for regenerative medicine applications and cell-based therapies.
As a widely used cell culture supplement, fetal bovine serum (FBS) harbor high content of growth, proliferation, and adhesion factors. However, high cost, bio-safety, possible xenogeneic agent transmission, finite accessible, and ethical controversy are major obstacles that discourage the use of this additive. Accordingly, novel alternatives have been proposed with various pros and cons. Still, caution should be taken in choosing suitable substitute given that the alteration in the main aspects of cultured cells can be biased the consequences of clinical applications. Herein, the authors evaluated the impact of cord blood serum harvesting by hydroxyethyl starch (CBS-HES), as an enriched source of growth factors, on the basic mesenchymal stem cells (MSCs) characteristics. In the present experiment, umbilical cord-derived MSCs were isolated and continuously nourished with Dulbecco’s Modified Eagle Medium containing either 10, 15, and 20% CBS-HES or FBSs to compare their morphology, immunophenotype, growth and proliferation rate, death rate, cell cycle, and gene expression profiles. Although all enriched media supported the expansion of MSCs with comparable morphology, cell surface markers, death rate, c-MYC and p16 expression, and growth rate, CBS-HES treated cells significantly (P < 0.05) expressed more hTERT gene in a concentration-dependent manner. Yet no significant shift was observed in the cell cycle of cultured cells using the same concentrations of additives, a finding which further confirmed by Ki-67 immunostaining. CBS-HES as an available and affordable additive, seems to be an optimal, relatively safe, and promising FBS alternative for cultivation, propagation, and subsequent clinical applications of MSCs.
