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.

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.

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.

Reduction of multiple pregnancy in ART.

It has been recognized that multiple pregnancy is associated with the increase risks to both maternal and newborn compared to singleton pregnancy. High incidence of premature birth complicated in the multiple pregnancy not only result in the high neonatal morbidity and mortality rate but also consume a high financial cost for the intensive neonatal care and may end up with the long-term childhood neurological handicaps. With the modern infertility treatment including ovarian hyperstimulation and assisted reproduction, on one hand has been resulted in a successful pregnancy rate but on the other hand has been consistency reported of the high incidence of multiple pregnancy especially high-order fetal pregnancy. The only way to prevent multiple pregnancy occurring from the infertility treatment is to induce monofollicular ovulation in non-IVF cycle and to do single embryo transfer in the IVF cycle but these have to be done without compromising the pregnancy rate. To achieve this goal, optimal IVF lab has to be developed which include optimal in vitro embryo culture system, effective embryo selection from the embryo pool for transfer and the efficiency of the cryopreservation of the surplus embryo for frozen-thaw cycle to augment the cumulative pregnancy rate. The doctor's responsibility to give information of the neonatal risk and cost of the multiple pregnancy, the patient's knowledge and attitude on the risks of multi-fetal pregnancy and the guideline for the ART practice are also essential to bring down the multiple births occurring from the treatment of the infertility.

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.