Karyotype In Culture Research Articles

Hypoxic Preconditioning Increases Survival and Pro-Angiogenic Capacity of Human Cord Blood Mesenchymal Stromal Cells In Vitro.

Hypoxic preconditioning was shown to improve the therapeutic efficacy of bone marrow-derived multipotent mesenchymal stromal cells (MSCs) upon transplantation in ischemic tissue. Given the interest in clinical applications of umbilical cord blood-derived MSCs, we developed a specific hypoxic preconditioning protocol and investigated its anti-apoptotic and pro-angiogenic effects on cord blood MSCs undergoing simulated ischemia in vitro by subjecting them to hypoxia and nutrient deprivation with or without preceding hypoxic preconditioning. Cell number, metabolic activity, surface marker expression, chromosomal stability, apoptosis (caspases-3/7 activity) and necrosis were determined, and phosphorylation, mRNA expression and protein secretion of selected apoptosis and angiogenesis-regulating factors were quantified. Then, human umbilical vein endothelial cells (HUVEC) were subjected to simulated ischemia in co-culture with hypoxically preconditioned or naïve cord blood MSCs, and HUVEC proliferation was measured. Migration, proliferation and nitric oxide production of HUVECs were determined in presence of cord blood MSC-conditioned medium. Cord blood MSCs proved least sensitive to simulated ischemia when they were preconditioned for 24 h, while their basic behavior, immunophenotype and karyotype in culture remained unchanged. Here, “post-ischemic” cell number and metabolic activity were enhanced and caspase-3/7 activity and lactate dehydrogenase release were reduced as compared to non-preconditioned cells. Phosphorylation of AKT and BAD, mRNA expression of BCL-XL, BAG1 and VEGF, and VEGF protein secretion were higher in preconditioned cells. Hypoxically preconditioned cord blood MSCs enhanced HUVEC proliferation and migration, while nitric oxide production remained unchanged. We conclude that hypoxic preconditioning protects cord blood MSCs by activation of anti-apoptotic signaling mechanisms and enhances their angiogenic potential. Hence, hypoxic preconditioning might be a translationally relevant strategy to increase the tolerance of cord blood MSCs to ischemia and improve their therapeutic efficacy in clinical applications.

Transient bursts of Zscan4 expression are accompanied by the rapid derepression of heterochromatin in mouse embryonic stem cells.

Mouse embryonic stem cells (mESCs) have a remarkable capacity to maintain normal genome stability and karyotype in culture. We previously showed that infrequent bursts of Zscan4 expression (Z4 events) are important for the maintenance of telomere length and genome stability in mESCs. However, the molecular details of Z4 events remain unclear. Here we show that Z4 events involve unexpected transcriptional derepression in heterochromatin regions that usually remain silent. During a Z4 event, we see rapid derepression and rerepression of heterochromatin leading to a burst of transcription that coincides with transient histone hyperacetylation and DNA demethylation, clustering of pericentromeric heterochromatin around the nucleolus, and accumulation of activating and repressive chromatin remodelling complexes. This heterochromatin-based transcriptional activity suggests that mESCs may maintain their extraordinary genome stability at least in part by transiently resetting their heterochromatin.

MODELING GENETIC Diseases through Reprogramming of HUMAN Amniotic Liquid Derived CELLS

AbstractAbstract 4789Pluripotency and self-renewal, two key characteristics of induced pluripotent stem cells (IPS), make these cells ideally suited for modeling diseases in vitro and generating biological resources usable for drug screening and cell therapy. However, the reprogramming efficiency of somatic cells greatly varies according to the cell type, to the in vitro proliferation index, the number of passages and the age of the donor. Human amniotic liquid-derived cells (hALDC), collected during amniocentesis for the prenatal diagnosis of genetic diseases, represent an abundant source of primary cells. In preliminary experiments we have shown that hALDC expressed endogenous Oct4 and Sox2 proteins suggesting that could be readily amenable to reprogramming. To this end, we have used two strategies using either hALDC or neonatal fibroblasts: (1) lentivirus mediated gene transfer of OCT4, SOX2, LIN28, NANOG, (2) retroviruses mediated gene transfer of OCT4, SOX2, CMYC, KLF4 and (3) lentiviral transfer of OCT4, SOX2. hALDC transduced by these viruses were placed on MEF and b-FGF (10 ng/ml) with daily medium changes. One to three weeks after infection, typical human ES-like colonies could be picked up for expansion before being characterized. HALDC show an increased reprogramming potential with the [OCT4, SOX2, LIN28, NANOG] and [OCT4, SOX2] cocktails, when compared to reprogramming of neonatal fibroblasts. Twelve hALDC-derived-IPS cells were obtained from 12 different samples of amniotic fluid. All hALDC-IPS cell lines maintained a normal karyotype in culture and displayed the morphology and characteristics of human embryonic stem cells, including the surface expression of Tra-160, SSEA-3, SSEA-4, HESCA-1 and alkaline phosphatase, and formed multi-lineaged teratomas upon injection to NOD-SCID mice. Gene expression profiles of the IPS cell lines reveal a high correlation coefficient between hALDC-iPS cells and human embryonic stem cells, and a low correlation between hALDC-iPS and hALDC. When compared to hES cells H1, H9 and Cl01, these cell lines generated hematopoiesis with a variable efficiency in vitro.Amongst the hALDC-IPS cell lines generated by our laboratory (http://www.hescreg.eu/) four lines carry an inherited trisomy of chromosome 21, and three lines carry the homozygous “S” mutation in the beta-globin gene of sickle-cell anemia. All hALDC-IPS cell are currently banked at the Human Pluripotent Stem Cell Core Facility, France. In conclusion, hALDC can be rapidly and efficiently reprogrammed to pluripotency with a limited number of transgenes. Moreover, hALDC-IPS cell lines derived from patients can be used to modelize in vitro the phenotypic features of monogenic diseases such as sickle cell anemia or more complex, multifactorial disorders such as Down's syndrom. The ability to generate hematopoietic differentiation from these cell lines will facilitate the modelling of these hematopoietic disorders. Disclosures:No relevant conflicts of interest to declare.

An XX male: cytogenetic and endocrine studies.

A 3 year old black male with ambiguous genitalia had a 46, XY karyotype in a bone marrow culture and an intermediate buccal smear result, suggestive of a mosaic of chromatin positive and chromatin negative cells. Upon re-evaluation at age 15 years, he has a 30% positive buccal smear and a 46, XX karyotype in cultures of peripheral blood lymphocytes, skin fibroblasts, bone marrow, and testis. No Y-body fluorescence was detectable in interphase cells from the testicular biopsy or the various cultures. The testicular biopsy appeared similar to that of XXY males, and primary hypogonadism was documented by elevated LH (107 mIU/ml) and FSH (57 mIU/ml) levels in conjunction with low testosterone (142 ng/100 ml). Administration of hCG produced qualitatively normal acute responses of testosterone and estrogens. The cytogenetic data provide support for the theory that at least some XX males once had a Y-containing cell line which was subsequently lost.