Carcinogenicity, efficiency and biosafety analysis in xeno-free human amniotic stem cells for regenerative medical therapies.

BACKGROUND AIMS: Human amniotic mesenchymal stromal cells (hAMSCs) are a potent and attractive stem cell source for use in regenerative medicine. However, the safe uses of therapeutic-grade MSCs are equally as important as the efficiency of MSCs. To provide efficient, clinic-compliant (safe for therapeutic use) MSCs, hAMSC lines that completely eliminate the use of animal products and have been characterized for carcinogenicity and biosafety are required. METHODS: Here, we have efficiently generated 10 hAMSC lines under human umbilical cord blood serum (hUCS)-supplemented medium (xeno-free culture) and fetal bovine serum (FBS)-supplemented medium (standard culture) and investigated carcinogenicity and immunosuppressive properties in the resultant hAMSC lines. All hAMSC lines were examined for efficiency (growth kinetics, cryopreservation, telomere length, phenotypic characterization, differentiation potential), carcinogenicity (proto-oncogene and tumor suppressor gene and epigenomic stability) and safety (immunosuppressive properties). RESULTS: Stem cell characteristics between the xeno-free hAMSC lines and the cell lines generated using the standard culture system showed no differences. Xeno-free hAMSC lines displayed normal growth proliferation potential, morphological, karyotypic, phenotypic differentiation properties and telomere lengths. Additionally, they retained normal immunosuppressive effects. As a marker of carcinogenicity and biosafety, proto-oncogenes expression levels showed no differences in xeno-free hAMSCs, and we detected no SNP mutations on hotspot codons of the P53 tumor suppressor gene and stable epigenomic imprinting in xeno-free hAMSC lines. CONCLUSIONS: Xeno-free hAMSC lines retain essential stem cell characteristics, with a high degree of certainty for meeting biosafety and carcinogenicity standards for a xeno-free system supplemented with allogenic hUCS. The cell lines are suitable and valuable for therapeutic purposes.

A xeno-free culture method that enhances Wharton's jelly mesenchymal stromal cell culture efficiency over traditional animal serum-supplemented cultures.

BACKGROUND AIMS: Mesenchymal stromal cell (MSC) transplantation holds great promise for use in medical therapies. Several key features of MSCs, including efficient cell growth, generation of sufficient cell numbers and safety, as determined by teratoma formation, make MSCs an ideal candidate for clinical use. However, MSCs derived under standard culture conditions, co-cultured with animal by-products, are inappropriate for therapy because of the risks of graft rejection and animal virus transmission to humans. Alternative serum sources have been sought for stem cell production. METHODS: We demonstrate for the first time that human serum from umbilical cord blood (hUCS) is an effective co-culture reagent for MSC production from Wharton's jelly MSCs (WJMSCs). Ten umbilical cords were used to generate parallel cultures of WJMSC lines under medium supplemented with hUCS and embryonic stem cell-qualified fetal bovine serum. The WJMSC lines from each medium were analyzed and compared with regard to cell line derivation, proliferation ability and characteristic stability. RESULTS: The phenotypic characteristics of WJMSC derived under either medium showed no differences. WJMSC lines derived under hUCS medium displayed comparable primary culture cell outgrowth, lineage differentiation capacity and cell recovery after cryopreservation compared with supplementation with embryonic stem cell-qualified fetal bovine serum medium. However, superior cell proliferation rates and retention of in vitro propagation (>22 passages) were observed in WJMSC cultures supplemented with hUCS. Additionally, more robust population doubling times were observed in hUCS-supplemented cultures. CONCLUSIONS: We conclude that hUCS is an efficient and effective serum source for animal product-free WJMSC line production and can generate MSC lines that may be appropriate for therapeutic use.

MSC secretome from amniotic fluid halts IL-1ß and TNF-α inflammation via the ERK/MAPK pathway, promoting cartilage regeneration in OA in vitro.

Osteoarthritis (OA) is a degenerative disease that causes chronic pain and disability worldwide. This disease is mainly caused by IL-1ß and TNF-α, which lead to cartilage degradation and inhibit the repair capacity of damaged cartilage. Recent studies have shown that amniotic fluid mesenchymal stem cells (AF-MSCs) secrete proteins that can effectively help in the treatment of cartilage damaged by OA. However, the underlying mechanism is still unclear. Therefore, the aim of this study was to investigate the effects and mechanisms behind the healing properties of the AF-MSC secretome (AFS-se) under OA conditions. This study involved growing chondrocyte progenitor cells (CPCs) and traumatized cartilage tissues in the presence of the cytokines IL-1ß and TNF-α, which mimic OA conditions. AFS-se was then added to the culture medium to determine its effect on the CPCs and cartilage. Cell migration, endogenous cell outgrowth, the expression of chondrogenic and anabolic genes, and the mechanism of proteins in the NF-κB and MAPK signaling pathways were examined in this study. AFS-se inhibited the inflammatory effects of IL-1ß and TNF-α by significantly reducing ERK phosphorylation in the MAPK signaling pathway and decreasing downstream proinflammatory COX2 products. The impaired CPCs recovered their ability to migrate, and endogenous CPCs in injured osteoarthritic cartilage were able to regrow in response to inflammatory stimuli. Additionally, the expression of anabolic genes such as Col I, Col II, and IGF1 was restored in defective CPCs. In conclusion, this study demonstrated that AFS-se has therapeutic effects on OA by inhibiting the inflammatory functions of IL-1ß and TNF-α through protein phosphorylation in the MAPK pathway while also promoting the regenerative and self-repair functions of CPCs in traumatized cartilage.

Effects of vitrification cryoprotectant treatment and cooling method on the viability and development of buffalo oocytes after intracytoplasmic sperm injection.

In vitro matured (IVM) buffalo oocytes at the metaphase of the second meiotic division (MII) were vitrified in 20% Me(2)SO: 20% EG (v/v) and 0.5M sucrose (VA), or 35% EG (v/v), 50mg/mL polyvinylpyrrolidone (PVP), and 0.4M trehalose (VB), either on cryotops or as 2µL microdrops. The viability was assessed after warming by fluorescein diacetate (FDA) staining and all surviving oocytes were subjected to ICSI and ethanol activation. All vitrified groups had similar recovery rates but both VA groups had significantly higher survival and pronuclear formation rates than either of the VB groups. Non treated control oocytes and non cryopreserved oocytes exposed to FDA had significantly higher survival, 2nd polar body extrusion, PN and blastocyst formation rates than any of the four vitrified groups (P<0.05). In conclusion The cryotop and microdrop methods are equally effective for buffalo oocyte vitrification, and although vitrification in VA solution yielded higher rates of survival and formation of 2 pronuclei than VB, the rate of blastocyst formation was comparable for both solutions. A detailed analysis of oocytes that extruded the second polar body after ICSI and activation revealed that only a minority (7-20% of the vitrified and 46-48% of the control oocytes) also had two pronuclei, indicating that normal activation is compromised by vitrification.

Hypoxia-induced amniotic fluid stem cell secretome augments cardiomyocyte proliferation and enhances cardioprotective effects under hypoxic-ischemic conditions.

Secretome derived from human amniotic fluid stem cells (AFSC-S) is rich in soluble bioactive factors (SBF) and offers untapped therapeutic potential for regenerative medicine while avoiding putative cell-related complications. Characterization and optimal generation of AFSC-S remains challenging. We hypothesized that modulation of oxygen conditions during AFSC-S generation enriches SBF and confers enhanced regenerative and cardioprotective effects on cardiovascular cells. We collected secretome at 6-hourly intervals up to 30 h following incubation of AFSC in normoxic (21%O2, nAFSC-S) and hypoxic (1%O2, hAFSC-S) conditions. Proliferation of human adult cardiomyocytes (hCM) and umbilical cord endothelial cells (HUVEC) incubated with nAFSC-S or hAFSC-S were examined following culture in normoxia or hypoxia. Lower AFSC counts and richer protein content in AFSC-S were observed in hypoxia. Characterization of AFSC-S by multiplex immunoassay showed higher concentrations of pro-angiogenic and anti-inflammatory SBF. hCM demonstrated highest proliferation with 30h-hAFSC-S in hypoxic culture. The cardioprotective potential of concentrated 30h-hAFSC-S treatment was demonstrated in a myocardial ischemia-reperfusion injury mouse model by infarct size and cell apoptosis reduction and cell proliferation increase when compared to saline treatment controls. Thus, we project that hypoxic-generated AFSC-S, with higher pro-angiogenic and anti-inflammatory SBF, can be harnessed and refined for tailored regenerative applications in ischemic cardiovascular disease.

A novel method to derive amniotic fluid stem cells for therapeutic purposes.

BACKGROUND: Human amniotic fluid stem (hAFS) cells have become an attractive stem cell source for medical therapy due to both their ability to propagate as stem cells and the lack of ethical debate that comes with the use of embryonic stem cells. Although techniques to derive stem cells from amniotic fluid are available, the techniques have limitations for clinical uses, including a requirement of long periods of time for stem cell production, population heterogeneity and xeno-contamination from using animal antibody-coated magnetic beads. Herein we describe a novel isolation method that fits for hAFS derivation for cell-based therapy. METHODS AND RESULTS: With our method, single hAFS cells generate colonies in a primary culture of amniotic fluid cells. Individual hAFS colonies are then expanded by subculturing in order to make a clonal hAFS cell line. This method allows derivation of a substantial amount of a pure stem cell population within a short period of time. Indeed, 108 cells from a clonal hAFS line can be derived in two weeks using our method, while previous techniques require two months. The resultant hAFS cells show a 2-5 times greater proliferative ability than with previous techniques and a population doubling time of 0.8 days. The hAFS cells exhibit typical hAFS cell characteristics including the ability to differentiate into adipogenic-, osteogenic- and neurogenic lineages, expression of specific stem cell markers including Oct4, SSEA4, CD29, CD44, CD73, CD90, CD105 and CD133, and maintenance of a normal karyotype over long culture periods. CONCLUSIONS: We have created a novel hAFS cell derivation method that can produce a vast amount of high quality stem cells within a short period of time. Our technique makes possibility for providing autogenic fetal stem cells and allogeneic cells for future cell-based therapy.

P53 Mutation and Epigenetic Imprinted IGF2/H19 Gene Analysis in Mesenchymal Stem Cells Derived from Amniotic Fluid, Amnion, Endometrium, and Wharton's Jelly.

Mesenchymal stem cells (MSC) are promising cells for medical therapy. In in vitro expansion, MSC can give rise to progeny with genomic and epigenomic alterations, resulting in senescence, loss of terminal differentiation, and transformation to cancer. However, MSC genome protects its genetic instability by a guardian function of the P53 tumor suppressor gene and epigenetic balance system during MSC culture. Mutations of P53 and epigenetic alterations have been reported to disrupt the quality and quantity of MSC and initiate tumorigenesis. We monitor P53 and epigenetic changes in MSC derived from amniotic fluid (AF-MSC), amnion membrane (AM-MSC), endometrium (EM-MSC), and Wharton's jelly (WJ-MSC) by the missense mutation analysis of the P53 gene and the expression levels of P53, and epigenetic insulin-like growth factor 2 (IGF2) and H19-imprinted genes. Our work demonstrates a variation of P53 expression among different MSC types. AF-MSC has a high P53 expression level with retaining a stability of P53 expression throughout a long culture period, whereas EM-MSC and WJ-MSC showed variation of P53 gene expression during culture. Epigenetic analysis showed a stable H19 expression pattern in AF-MSC, AM-MSC, and EM-MSC culture, whereas H19 expression fluctuated in WJ-MSC culture. We conclude that gene instability can be found during in vitro MSC expansion. With awareness to MSC quality and safety in MSC transformation risk, P53 mutation and IGF2 and H19-imprinted gene analysis should be applied to monitor in therapeutic-grade MSC. We also demonstrated that AF-MSC is one of the most interesting MSC for medical therapy because of its high genomic stability and epigenetic fidelity.

Human Umbilical Cord Blood-Derived Serum for Culturing the Supportive Feeder Cells of Human Pluripotent Stem Cell Lines.

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 coculture with hPSCs. The use of FBS might limit the clinical application of hPSCs. Recently, human cord blood-derived serum (hUCS) showed a positive effect on culture of mesenchymal stem cells. It is interesting to test whether hUCS can be used for culture of feeder cells of hPSCs. This study was aimed to replace FBS with hUCS for culturing the human foreskin fibroblasts (HFFs) prior to feeder cell preparation. The results showed that HFFs cultured in hUCS-containing medium (HFF-hUCS) displayed fibroblastic features, high proliferation rates, short population doubling times, and normal karyotypes after prolonged culture. Inactivated HFF-hUCS expressed important genes, including Activin A, FGF2, and TGFß1, which have been implicated in the maintenance of hPSC pluripotency. Moreover, hPSC lines maintained pluripotency, differentiation capacities, and karyotypic stability after being cocultured for extended period with inactivated HFF-hUCS. Therefore, the results demonstrated the benefit of hUCS for hPSCs culture system.

Successful derivation of xeno-free mesenchymal stem cell lines from endometrium of infertile women.

Transplantation of mesenchymal stem cells (MSC) can effectively repair endometrial deficiencies, including infertile patients with a problem of inadequate endometrium thickness. Although, MSC derived from different organ sources have a similarity of MSC specific characteristics, endometrial stem cells (EMSC) are temporally regulated throughout the menstrual cycle in a micro-environmental niche found only in endometrial tissue. Given the micro-environment niche, developing treatments for endometrial disorders with EMSC should be a top priority. To provide EMSC that afford safety for therapeutic usage, we have established a completely xeno-free EMSC line derivation protocol using human allogenic umbilical cord serum instead of animal derived reagents, and proved that it is feasible to generate xeno-free EMSC lines from infertile patient donors using these conditions. Our results demonstrate the successful derivation of xeno-free EMSC lines from 10 out of 10 infertile patients. The resultant xeno-free EMSC lines showed typical MSC morphology, phenotypic markers, differentiation capacity, telomere length and normal karyotypes. They showed superior proliferation capability, but lower expression of proto-oncogenes, to the lines generated under standard (animal derived reagents) culture. Biosafety of xeno-free EMSC lines also displayed in retention of immunosuppressive ability, epigenetic stability by imprinted genes expression, proto-oncogenes expression and no mutation of specific codon on p53 tumor suppressor gene. Taken together, these data indicate that our cells may be safe for clinical use. In conclusion, we have succeeded in establishing completely xeno-free EMSC lines and demonstrate for the first time that autogenic and xeno-free EMSC lines can be generated from infertile women.

Potential role of N-benzylcinnamide in inducing neuronal differentiation from human amniotic fluid mesenchymal stem cells.

Neurodegenerative disorders are characterized by chronic and progressive loss of neurons in structure and function related to aging, such as Alzheimer's disease, the latter characterized by the degeneration of cholinergic neurons in basal forebrain connected to the cerebral cortex and hippocampus. Amniotic fluid mesenchymal stem cells (AF-MSCs) have been proposed as one of the candidates for stem cell therapy of nervous system disorders. This study demonstrates that incubation of AF-MSCs, obtained from 16 to 20 week pregnant women, with 10ng/ml bone morphogenetic protein (BMP)-9 for 48h in conditioned medium resulted in transdifferentiation to cholinergic neuronal-like cells. This phenomenon could also be obtained with N-benzylcinnamide (PT-3). Pre-treatment for 1h with 10nM PT-3 augmented BMP-9 transdifferentiation effect, elevated ßIII-tubulin cell numbers and fluorescence intensity of immunoreactive ChAT, ameliorated BMP-9-related production of reactive oxygen species and enhanced anti-apoptosis status of the neuronal-like cells. The transdiffirentiation process was accompanied by increased p53 but decreased Notch1 and SIRT1 (p53 deacetylase) levels, and activation of p38, ERK1/2 MAPK, and PI3K/Akt pathways, in concert with inactivation of JNK, all of which were accentuated by PT-3 pre-treatment. These findings suggest that N-benzylcinnamide may provide a useful adjuvant in BMP-9-induced transdifferentiation of AFMSCs into ultimately cholinergic neurons.

Epigenetic analysis and suitability of amniotic fluid stem cells for research and therapeutic purposes.

Amniotic fluid stem cells (AFSs) are interesting mesenchymal stem cells (MSCs) that are characterized by their great potential for cell proliferation and differentiation compared with other types of MSCs identified to date. However, MSCs in prolonged culture have been found to exhibit defects in genetic stability and differentiation capacity. Epigenetic anomalies have been hypothesized to be a cause of these defects. Here, we investigated the genomic methylation and genetic imprinting in AFSs during prolonged in vitro culture. Four human imprinted genes, insulin-like growth factor 2 (IGF2), H19, small nuclear ribonucleoprotein polypeptide N gene (SNRPN), and mesoderm-specific transcript (MEST), were evaluated for their expression levels and methylation statuses in AFS lines. The data revealed epigenetic instability in high passage number AFS cultures. The real-time polymerase chain reaction analysis showed that the expression levels of the imprinted genes gradually increased with increased time in culture. The loss of parental allele-specific imprinting for at least 1 gene among IGF2, H19, and SNRPN was observed in every AFS line after passage 8 using allelic expression analysis. The imprinting control regions (ICRs) of the IGF2 and H19 genes were assayed for site-specific methylation using bisulfite sequencing. This assay revealed a variable level of methylated CpG sites in the ICRs of IGF2 and H19. This variable level of CpG methylation is related to the aberrant expression of the IGF2 and H19 genes in late-passage AFSs. Our results did not reveal any irregularity in the epigenetic control system in the early-passage AFSs, indicating that the standard in vitro culturing of AFSs used in medical treatments should be limited to 8 passages.

Cryopreservation of immature buffalo oocytes: effects of cytochalasin B pretreatment on the efficiency of cryotop and solid surface vitrification methods.

The aim of the present study was to compare the efficiency of the solid surface (SSV), cryotop (CT) vitrification methods and cytochalasin B (CB) pretreatment for cryopreservation of immature buffalo oocytes. Cumulus-oocyte complexes (COCs) were placed for 1 min in TCM199 containing 10% dimethylsulfoxide (DMSO), 10% ethylene glycol (EG), and 20% fetal bovine serum, and then transferred for 30 s to base medium containing 20% DMSO, 20% EG and 0.5 mol/L sucrose. CB pretreated ((+)CB) or non-pretreated ((-)CB) COCs were vitrified either by SSV or CT. Surviving vitrified COCs were selected for in vitro maturation (IVM) and in vitro fertilization (IVF). The rate of viable oocytes after vitrification in CT groups (82%) was significantly lower (P < 0.05) than that in a fresh control group (100%), but significantly higher (P < 0.05) than those in SSV groups (71-72%). Among vitrified groups, the highest maturation rate was obtained in the CT (-)CB group (32%). After IVF, the cleavage and blastocyst formation rates were similar among vitrified groups but significantly lower than those of the control group. In conclusion, a higher survival rate of oocytes after vitrification and IVM was obtained in the CT group compared with that in the SSV group, indicating the superiority of the CT method. Pretreatment with CB did not increase the viability, maturation or embryo development of vitrified oocytes.

Effect of donor cell types on developmental potential of cattle (Bos taurus) and swamp buffalo (Bubalus bubalis) cloned embryos.

This study investigated the effect of donor cell types on the developmental potential and quality of cloned swamp buffalo embryos in comparison with cloned cattle embryos. Fetal fibroblasts (FFs), ear fibroblasts (EFs), granulosa cells (GCs) and cumulus cells (CCs) were used as the donor cells in both buffalo and cattle. The cloned cattle or buffalo embryos were produced by fusion of the individual donor cells with enucleated cattle or buffalo oocytes, respectively. The reconstructed (cloned) embryos and in vitro matured oocytes without enucleation were parthenogenetically activated (PA) and cultured for 7 days. Their developmental ability to the blastocyst stage was evaluated. The total number of trophectoderm (TE) and inner cell mass (ICM) cells and the ICM ratio in each blastocyst was determined by differential staining as an indicator of embryo quality. The fusion rate of CCs with enucleated oocytes was significantly lower than for those of other donor cell types both in cattle and buffalo. The rates of cleavage and development to the 8-cell, morula and blastocyst stages of cloned embryos derived from all donor cell types did not significantly differ within the same species. However, the cleavage rate of cloned cattle embryos derived from FFs was significantly higher than those of cattle PA and cloned buffalo embryos. The blastocyst rates of cloned cattle embryos, except for the ones derived from CCs, were significantly higher than those of cloned buffalo embryos. In buffalo, only cloned embryos derived from CCs showed a significantly higher blastocyst rate than that of PA embryos. In contrast, all the cloned cattle embryos showed significantly higher blastocyst rates than that of PA embryos. There was no difference in ICM ratio among any of the blastocysts derived from any of the donor cell types and PA embryos in both species. FFs, EFs, GCs and CCs had similar potentials to support development of cloned cattle and buffalo embryos to the blastocyst stage with the same quality.