Hanna Segev

Human feeder layers for human embryonic stem cells.

Abstract Human embryonic stem (hES) cells hold great promise for future use in various research areas, such as human developmental biology and cell-based therapies. Traditionally, these cells have been cultured on mouse embryonic fibroblast (MEF) feeder layers, which permit continuous growth in an undifferentiated stage. To use these unique cells in human therapy, an animal-free culture system must be used, which will prevent exposure to mouse retroviruses. Animal-free culture systems for hES cells enjoy three major advantages in the basic culture conditions: 1) the ability to grow these cells under serum-free conditions, 2) maintenance of the cells in an undifferentiated state on Matrigel matrix with 100% MEF-conditioned medium, and 3) the use of either human embryonic fibroblasts or adult fallopian tube epithelial cells as feeder layers. In the present study, we describe an additional animal-free culture system for hES cells, based on a feeder layer derived from foreskin and a serum-free medium. In this culture condition, hES cells maintain all embryonic stem cell features (i.e., pluripotency, immortality, unlimited undifferentiated proliferation capability, and maintenance of normal karyotypes) after prolonged culture of 70 passages (>250 doublings). The major advantage of foreskin feeders is their ability to be continuously cultured for more than 42 passages, thus enabling proper analysis for foreign agents, genetic modification such as antibiotic resistance, and reduction of the enormous workload involved in the continuous preparation of new feeder lines.

Differentiation of Human Embryonic Stem Cells into Insulin-Producing Clusters

Type I diabetes mellitus is caused by an autoimmune destruction of the insulin‐producing β cells. The major obstacle in using transplantation for curing the disease is the limited source of insulin‐producing cells. The isolation of human embryonic stem (hES) cells introduced a new prospect for obtaining a sufficient number of β cells for transplantation.

Enhanced reprogramming and cardiac differentiation of human keratinocytes derived from plucked hair follicles, using a single excisable lentivirus.

Induced pluripotent stem cells (iPSCs) represent an ideal cell source for future cell therapy and regenerative medicine. However, most iPSC lines described to date have been isolated from skin fibroblasts or other cell types that require harvesting by surgical intervention. Because it is desirable to avoid such intervention, an alternative cell source that can be readily and noninvasively isolated from patients and efficiently reprogrammed, is required. Here we describe a detailed and reproducible method to derive iPSCs from plucked human hair follicle keratinocytes (HFKTs). HFKTs were isolated from single plucked hair, then expanded and reprogrammed by a single polycistronic excisable lentiviral vector. The reprogrammed HFKTs were found to be very sensitive to human embryonic stem cell (hESC) growth conditions, generating a built-in selection with easily obtainable and very stable iPSCs. All emerging colonies were true iPSCs, with characteristics typical of human embryonic stem cells, differentiated into derivatives of all three germ layers in vitro and in vivo. Spontenaeouly differentiating functional cardiomyocytes (CMs) were successfully derived and characterized from these HFKT-iPSCs. The contracting CMs exhibited well-coordinated intracellular Ca²+ transients and contractions that were readily responsive to β-adrenergic stimulation with isoproterenol. The introduction of Cre-recombinase to HFKT-iPSC clones was able to successfully excise the integrated vector and generate transgene-free HFKT-iPSC clone that could be better differentiated into contracting CMs, thereby revealing the desired cells for modeling human diseases. Thus, HFKTs are easily obtainable, and highly reprogrammed human cell source for all iPSC applications.

Molecular analysis of cardiomyocytes derived from human embryonic stem cells

During early embryogenesis, the cardiovascular system is the first system to be established and is initiated by a process involving the hypoblastic cells of the primitive endoderm. Human embryonic stem (hES) cells provide a model to investigate the early developmental stages of this system. When removed from their feeder layer, hESC create embryoid bodies (EB) which, when plated, develop areas of beating cells in 21.5% of the EB. These spontaneously contracting cells were demonstrated using histology, immunostaining and reverse transcription–polymerase chain reaction (RT‐PCR), to possess morphological and molecular characteristics consistent with cardiomyocytic phenotypes. In addition, the expression pattern of specific cardiomyocytic genes in human EB (hEB) was demonstrated and analyzed for the first time. GATA‐4 is the first gene to be expressed in 6‐day‐old EB. Alpha cardiac actin and atrial natriuretic factor are expressed in older hEB at 10 and 20 days, respectively. Light chain ventricular myosin (MLC‐2V) was expressed only in EB with beating areas and its expression increased with time. Alpha heavy chain myosin (α‐MHC) expression declined in the pulsating hEB with time, in contrast to events in EB derived from mice. We conclude that human embryonic stem cells can provide a useful tool for research on embryogenesis in general and cardiovascular development in particular.

Targeting pancreatic progenitor cells in human embryonic stem cell differentiation for the identification of novel cell surface markers.

New sources of beta cells are needed in order to develop cell therapies for patients with diabetes. An alternative to forced expansion of post-mitotic beta cells is the induction of differentiation of stem-cell derived progenitor cells that have a natural self-expansion capacity into insulin-producing cells. In order to learn more about these progenitor cells at different stages along the differentiation process in which they become progressively more committed to the final beta cell fate, we took the approach of identifying, isolating and characterizing stage specific progenitor cells. We generated human embryonic stem cell (HESC) clones harboring BAC GFP reporter constructs of SOX17, a definitive endoderm marker, and PDX1, a pancreatic marker, and identified subpopulations of GFP expressing cells. Using this approach, we isolated a highly enriched population of pancreatic progenitor cells from hESCs and examined their gene expression with an emphasis on the expression of stage-specific cell surface markers. We were able to identify novel molecules that are involved in the pancreatic differentiation process, as well as stage-specific cell markers that may serve to define (alone or in combination with other markers) a specific pancreatic progenitor cell. These findings may help in optimizing conditions for ultimately generating and isolating beta cells for transplantation therapy.

Subcloning and Alternative Methods for the Derivation and Culture of Human Embryonic Stem Cells

Human embryonic stem (ES) cell lines may have broad applications, including the study of development and the differentiation process, lineage commitment, self-maintenance, and precursor cell maturation. They may also serve as models in research done on the functions of genes and proteins, drug testing, and drug toxicity. The first human ES cells were derived by Thomson and colleagues (1) from the inner cell mass (ICM) of surplus blastocysts donated by couples undergoing in vitro fertilization treatments. These lines met most of the criteria for ES cell lines listed in Table 1, but their clonality was not tested in that study. Also, the ability of human ES cells to contribute to embryonic development in chimeric embryos cannot be examined for obvious ethical reasons. Since the first report on human ES cell derivation, several other groups have reported the derivation of additional lines (2–4) At present, there are more than 70 human ES cell lines in several laboratories around the world, according to a list published by the National Institutes of Health (NIH; http://www.nih.gov/news/stemcell/index/news/stemcell/index). Although the NIH list does not offer full information on all the lines fulfilling all the ES cell criteria listed in Table 1, it suggests that the derivation of human ES cells is a reproducible procedure with reasonable success rates.