Human Oral Mucosa as a Potentially Effective Source of Neural Crest Stem Cells for Clinical Practice.

We report in this study on the isolation and expansion of neural crest stem cells (NCSCs) from the epithelium of oral mucosa (OM) using reagents that are GMP-certified and FDA-approved for clinical use. Characterization analysis showed that the levels of keratins K2, K6C, K4, K13, K31, and K15-specific to OM epithelial cells-were significantly lower in the experimental NCSCs. While SOX10 was decreased with no statistically significant difference, the earliest neural crest specifier genes SNAI1/2, Ap2a, Ap2c, SOX9, SOX30, Pax3, and Twist1 showed a trend in increased expression in NCSCs. In addition, proteins of Oct4, Nestin and Noth1 were found to be greatly expressed, confirming NCSC multipotency. In conclusion, our study showed that the epithelium of OM contains NCSCs that can be isolated and expanded with clinical-grade reagents to supply the demand for multipotent cells required for clinical applications in regenerative medicine. Supported by Emmaus Medical Inc.

cG-CAOMECS-clinical-grade cultured autologous oral mucosal epithelial cell sheet.

The present study reports the feasibility and successful production of rabbit cG-CAOMECS, designed to reconstruct corneal epithelium of patients with bilateral limbal stem cell deficiency. To produce a safe, chemically defined and FDA compliant cG-CAOMECS, oral mucosal epithelial cells were isolated from a biopsy of rabbit buccal tissue and seeded on a cGMP-certified cell culture surface coated with GMP-grade extracellular matrix. A newly designed clinical-grade medium (KaFa™ medium) was utilized to carry out cell expansion. Detachment and harvesting of the produced cell sheet was accomplished using collagenase treatment. Live cell imaging and morphological analysis techniques were used to examine cell growth. Cells attached onto the surface and self-assembled into colony-forming units (CFUs). Microscopic examination showed that CFUs formed during the first 5 days, and basal monolayer cell sheet formed in less than 10 days. Cells expanded to form a multilayered epithelial cell sheet that was harvested after 17-19 days in culture. Immunostaining and Western blot analyses showed that deltaNp63 was expressed in the basal cells and K3/K12 was expressed in the apical cells, indicating the presence of corneal epithelial-like cells in the produced cell sheet. Adhesion molecules, E-cadherin, beta-catenin, and Cnx43 were also expressed and exhibited the epithelial integrity of the cell sheet. The expression of integrin-beta1 and beta4 confirmed that the collagenase treatment used for detaching and harvesting the cell sheet did not have adverse effects. Our results showed that the utilization of clinical-grade and FDA-approved reagents successfully supported the production of cG-CAMECS.

Maternal High Fat Diet Programs Male Mice Offspring Hyperphagia and Obesity: Mechanism of Increased Appetite Neurons via Altered Neurogenic Factors and Nutrient Sensor AMPK.

Maternal high-fat (HF) is associated with offspring hyperphagia and obesity. We hypothesized that maternal HF alters fetal neuroprogenitor cell (NPC) and hypothalamic arcuate nucleus (ARC) development with preferential differentiation of neurons towards orexigenic (NPY/AgRP) versus anorexigenic (POMC) neurons, leading to offspring hyperphagia and obesity. Furthermore, these changes may involve hypothalamic bHLH neuroregulatory factors (Hes1, Mash1, Ngn3) and energy sensor AMPK. Female mice were fed either a control or a high fat (HF) diet prior to mating, and during pregnancy and lactation. HF male newborns were heavier at birth and exhibited decreased protein expression of hypothalamic bHLH factors, pAMPK/AMPK and POMC with increased AgRP. As adults, these changes persisted though with increased ARC pAMPK/AMPK. Importantly, the total NPY neurons were increased, which was consistent with the increased food intake and adult fat mass. Further, NPCs from HF newborn hypothalamic tissue showed similar changes with preferential NPC neuronal differentiation towards NPY. Lastly, the role of AMPK was further confirmed with in vitro treatment of Control NPCs with pharmacologic AMPK modulators. Thus, the altered ARC development of HF offspring results in excess appetite and reduced satiety leading to obesity. The underlying mechanism may involve AMPK/bHLH pathways.

Aneuploidy induces profound changes in gene expression, proliferation and tumorigenicity of human pluripotent stem cells.

Human pluripotent stem cells (hPSCs) tend to acquire genomic aberrations in culture, the most common of which is trisomy of chromosome 12. Here we dissect the cellular and molecular implications of this trisomy in hPSCs. Global gene expression analyses reveal that trisomy 12 profoundly affects the gene expression profile of hPSCs, inducing a transcriptional programme similar to that of germ cell tumours. Comparison of proliferation, differentiation and apoptosis between diploid and aneuploid hPSCs shows that trisomy 12 significantly increases the proliferation rate of hPSCs, mainly as a consequence of increased replication. Furthermore, trisomy 12 increases the tumorigenicity of hPSCs in vivo, inducing transcriptionally distinct teratomas from which pluripotent cells can be recovered. Last, a chemical screen of 89 anticancer drugs discovers that trisomy 12 raises the sensitivity of hPSCs to several replication inhibitors. Together, these findings demonstrate the extensive effect of trisomy 12 and highlight its perils for successful hPSC applications.

Differentiation of human limbal-derived induced pluripotent stem cells into limbal-like epithelium.

Limbal epithelial stem cell (LESC) deficiency (LSCD) leads to corneal abnormalities resulting in compromised vision and blindness. LSCD can be potentially treated by transplantation of appropriate cells, which should be easily expandable and bankable. Induced pluripotent stem cells (iPSCs) are a promising source of transplantable LESCs. The purpose of this study was to generate human iPSCs and direct them to limbal differentiation by maintaining them on natural substrata mimicking the native LESC niche, including feederless denuded human amniotic membrane (HAM) and de-epithelialized corneas. These iPSCs were generated with nonintegrating vectors from human primary limbal epithelial cells. This choice of parent cells was supposed to enhance limbal cell differentiation from iPSCs by partial retention of parental epigenetic signatures in iPSCs. When the gene methylation patterns were compared in iPSCs to parental LESCs using Illumina global methylation arrays, limbal-derived iPSCs had fewer unique methylation changes than fibroblast-derived iPSCs, suggesting retention of epigenetic memory during reprogramming. Limbal iPSCs cultured for 2 weeks on HAM developed markedly higher expression of putative LESC markers ABCG2, ΔNp63α, keratins 14, 15, and 17, N-cadherin, and TrkA than did fibroblast iPSCs. On HAM culture, the methylation profiles of select limbal iPSC genes (including NTRK1, coding for TrkA protein) became closer to the parental cells, but fibroblast iPSCs remained closer to parental fibroblasts. On denuded air-lifted corneas, limbal iPSCs even upregulated differentiated corneal keratins 3 and 12. These data emphasize the importance of the natural niche and limbal tissue of origin in generating iPSCs as a LESC source with translational potential for LSCD treatment.

The in vitro survival of human monosomies and trisomies as embryonic stem cells.

Chromosomal aneuploidies are responsible for severe human genetic diseases. Aiming at creating models for such disorders, we have generated human embryonic stem cell (hESC) lines from pre-implantation genetic screened (PGS) embryos. The overall analysis of more than 400 aneuploid PGS embryos showed a similar risk of occurrence of monosomy or trisomy for any specific chromosome. However, the generation of hESCs from these embryos revealed a clear bias against monosomies in autosomes. Moreover, only specific trisomies showed a high chance of survival as hESC lines, enabling us to present another categorization of human aneuploidies. Our data suggest that chromosomal haploinsufficiency leads to lethality at very early stages of human development.

Human embryonic stem cells as models for aneuploid chromosomal syndromes.

Syndromes caused by chromosomal aneuploidies are widely recognized genetic disorders in humans and often lead to spontaneous miscarriage. Preimplantation genetic screening is used to detect chromosomal aneuploidies in early embryos. Our aim was to derive aneuploid human embryonic stem cell (hESC) lines that may serve as models for human syndromes caused by aneuploidies. We have established 25 hESC lines from blastocysts diagnosed as aneuploid on day 3 of their in vitro development. The hESC lines exhibited morphology and expressed markers typical of hESCs. They demonstrated long-term proliferation capacity and pluripotent differentiation. Karyotype analysis revealed that two-third of the cell lines carry a normal euploid karyotype, while one-third remained aneuploid throughout the derivation, resulting in eight hESC lines carrying either trisomy 13 (Patau syndrome), 16, 17, 21 (Down syndrome), X (Triple X syndrome), or monosomy X (Turner syndrome). On the basis of the level of single nucleotide polymorphism heterozygosity in the aneuploid chromosomes, we determined whether the aneuploidy originated from meiotic or mitotic chromosomal nondisjunction. Gene expression profiles of the trisomic cell lines suggested that all three chromosomes are actively transcribed. Our analysis allowed us to determine which tissues are most affected by the presence of a third copy of either chromosome 13, 16, 17 or 21 and highlighted the effects of trisomies on embryonic development. The results presented here suggest that aneuploid embryos can serve as an alternative source for either normal euploid or aneuploid hESC lines, which represent an invaluable tool to study developmental aspects of chromosomal abnormalities in humans.

Human embryonic stem cells from aneuploid blastocysts identified by pre-implantation genetic screening.

Human embryonic stem cells are derived from the inner cell mass of pre-implantation embryos. The cells have unlimited proliferation potential and capacity to differentiate into the cells of the three germ layers. Human embryonic stem cells are used to study human embryogenesis and disease modeling and may in the future serve as cells for cell therapy and drug screening. Human embryonic stem cells are usually isolated from surplus normal frozen embryos and were suggested to be isolated from diseased embryos detected by pre-implantation genetic diagnosis. Here we report the isolation of 12 human embryonic stem cell lines and their thorough characterization. The lines were derived from embryos detected to have aneuploidy by pre-implantation genetic screening. Karyotype analysis of these cell lines showed that they are euploid, having 46 chromosomes. Our interpretation is that the euploid cells originated from mosaic embryos, and in vitro selection favored the euploid cells. The undifferentiated cells exhibited long-term proliferation and expressed markers typical for embryonic stem cells such as OCT4, NANOG, and TRA-1-60. The cells manifested pluripotent differentiation both in vivo and in vitro. To further characterize the different lines, we have analyzed their ethnic origin and the family relatedness among them. The above results led us to conclude that the aneuploid mosaic embryos that are destined to be discarded can serve as source for normal euploid human embryonic stem cell lines. These lines represent various ethnic groups; more lines are needed to represent all populations.

Derivation of euploid human embryonic stem cells from aneuploid embryos.

Human embryonic stem cells (HESCs) are pluripotent cells derived from the inner cell mass of preimplantation embryos. In this study, to isolate new lines of HESCs, we used blastocyst-stage embryos diagnosed as aneuploid in preimplantation genetic screening (PGS). During in vitro fertilization treatments, PGS is widely applied to identify chromosomal aneuploidies, especially in cases of advanced maternal age. Embryos that are detected as carrying aneuploidies are destined to be discarded unless donated for research. From 74 fresh PGS-defined aneuploid embryos, we derived seven HESC lines. Most of the embryos were left to hatch spontaneously through the hole created for blastomere biopsy and further treated by immunosurgery. The seven HESC lines exhibited morphology and markers typical of HESCs and the capacity for long-term proliferation. The derived HESC lines manifested pluripotent differentiation potential both in vivo and in vitro. Surprisingly, karyotype analysis of the HESC lines that were derived from these aneuploid embryos showed that the cell lines carry a normal euploid karyotype. We show that the euploidy was not achieved through chromosome duplication. Alternatively, we suggest that the euploid HESC lines originated from mosaic embryos consisting of aneuploid and euploid cells, and in vitro selection occurred to favor euploid cells. We assume that aneuploid HESC lines could be isolated mostly from embryos that are uniform for the aneuploidy. These results led us to conclude that the aneuploid mosaic embryos that are destined to be discarded can serve as an alternative source for normal euploid HESC lines.