Sharon Gerecht-Nir

Controlled, scalable embryonic stem cell differentiation culture.

Embryonic stem (ES) cells are of significant interest as a renewable source of therapeutically useful cells. ES cell aggregation is important for both human and mouse embryoid body (EB) formation and the subsequent generation of ES cell derivatives. Aggregation between EBs (agglomeration), however, inhibits cell growth and differentiation in stirred or high‐cell‐density static cultures. We demonstrate that the agglomeration of two EBs is initiated by E‐cadherin‐mediated cell attachment and followed by active cell migration. We report the development of a technology capable of controlling cell‐cell interactions in scalable culture by the mass encapsulation of ES cells in size‐specified agarose capsules. When placed in stirred‐suspension bioreactors, encapsulated ES cells can be used to produce scalable quantities of hematopoietic progenitor cells in a controlled environment.

Human Embryonic Stem Cells as an In Vitro Model for Human Vascular Development and the Induction of Vascular Differentiation

Early embryonic blood vessels are typically composed of fragile tubes of endothelial cells encircled by vascular smooth muscle cells. Early human vasculogenesis was explored in spontaneous and directed differentiation models derived from human embryonic stem (HES) cells. In a 3-dimensional (3D) model, HES cells were studied for their potential for vascular differentiation during the spontaneous formation of embryoid bodies. Directed differentiation was investigated by means of a 2-dimensional (2D) differentiation method to promote vascular differentiation from HES cells (without the formation of embryoid bodies). Using this latter approach, up-regulation of early lineage markers of endothelial progenitors were induced. Additional culture under strict conditions and exposure to angiogenic growth factors resulted in a prolonged differentiation pathway into mature endothelial cells and up-regulation of vascular smooth muscle cell markers. The use of 3D collagen gels and Matrigel assays for the induction and inhibition of human vascular sprouting in vitro further established the vascular potential of the cells generated by the 2D differentiation system. Our study shows that HES cells can provide useful models to study early differentiation and development of blood vessels. Moreover, the 2D differentiation model facilitates both the production of vascular lineage cells from HES cells for various potential therapeutic applications and also provides a model for studying the mechanisms involved in early human embryonic blood vessel development.

Human embryonic stem cells: A potential source for cellular therapy

Many degenerative human diseases reflect damage to cells that are not normally repaired or replaced, such as diabetes, Parkinsons disease, hepatic failure and congestive heart failure. Preliminary studies in animals and humans have suggested that these diseases may be treatable by transplantation of healthy cells. Such cells may be obtained by in vitro culture of embryonic stem cells, which are capable of differentiating into many cell types. This review discusses applicative approaches for the derivation, maintenance and safety of human embryonic stem (hES) cells as well as ethical concerns surrounding their possible source for cellular therapy. hES cells offer broad application in cellular therapy; however, this review specifically emphasizes on cardiovascular repair, generation and characterization of hES cell‐derived cardiomyocytes, vascular progenitors and differentiation of derivatives.

Design principle of gene expression used by human stem cells: implication for pluripotency

Human embryonic stem cells (ESC) are undifferentiated and are endowed with the capacities of self‐renewal and pluripotential differentiation. Adult stem cells renew their own tissue, but whether they can transdifferentiate to other tissues is still controversial. To understand the genetic program that underlies the pluripotency of stem cells, we compared the transcription profile of ESC with that of progenitor/stem cells of human hematopoietic and keratinocytic origins, along with their mature cells to be viewed as snapshots along tissue differentiation. ESC gene profiles show higher complexity with significantly more highly expressed genes than adult cells. We hypothesize that ESC use a strategy of expressing genes that represent various differentiation pathways and selection of only a few for continuous expression upon differentiation to a particular target. Such a strategy may be necessary for the pluripotency of ESC. The progenitors of either hematopoietic or keratinocytic cells also follow the same design principle. Using advanced clustering, we show that many of the ESC expressed genes are turned off in the progenitors/stem cells followed by a further down‐regulation in adult tissues. Concomitantly, genes specific to the target tissue are up‐regulated toward mature cells of skin or blood.

Vascular gene expression and phenotypic correlation during differentiation of human embryonic stem cells.

The study of the cascade of events of induction and sequential gene activation that takes place during human embryonic development is hindered by the unavailability of postimplantation embryos at different stages of development. Spontaneous differentiation of human embryonic stem cells (hESCs) can occur by means of the formation of embryoid bodies (EBs), which resemble certain aspects of early embryos to some extent. Embryonic vascular formation, vasculogenesis, is a sequential process that involves complex regulatory cascades. In this study, changes of gene expression along the development of human EBs for 4 weeks were studied by large‐scale gene screening. Two main clusters were identified—one of down‐regulated genes such as POU5, NANOG, TDGF1/Cripto (TDGF, teratocarcinoma‐derived growth factor‐1), LIN28, CD24, TERF1 (telomeric repeat binding factor‐1), LEFTB (left–right determination, factor B), and a second of up‐regulated genes such as TWIST, WNT5A, WT1, AFP, ALB, NCAM1. Focusing on the vascular system development, genes known to be involved in vasculogenesis and angiogenesis were explored. Up‐regulated genes include vasculogenic growth factors such as VEGFA, VEGFC, FIGF (VEGFD), ANG1, ANG2, TGFβ3, and PDGFB, as well as the related receptors FLT1, FLT4, PDGFRB, TGFβR2, and TGFβR3, other markers such as CD34, VCAM1, PECAM1, VE‐CAD, and transcription factors TAL1, GATA2, and GATA3. The reproducibility of the array data was verified independently and illustrated that many genes known to be involved in vascular development are activated during the differentiation of hESCs in culture. Hence, the analysis of the vascular system can be extended to other differentiation pathways, allocating human EBs as an in vitro model to study early human development. Developmental Dynamics 232:487–497, 2005.

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.

Vascular Development in Early Human Embryos and in Teratomas Derived from Human Embryonic Stem Cells

Abstract During early human embryonic development, blood vessels are stimulated to grow, branch, and invade developing tissues and organs. Pluripotent human embryonic stem cells (hESCs) are endowed with the capacity to differentiate into cells of blood and lymphatic vessels. The present study aimed to follow vasculogenesis during the early stages of developing human vasculature and to examine whether human neovasculogenesis within teratomas generated in SCID mice from hESCs follows a similar course and can be used as a model for the development of human vasculature. Markers and gene profiling of smooth muscle cells and endothelial cells of blood and lymphatic vessels were used to follow neovasculogenesis and lymphangiogenesis in early developing human embryos (4–8 weeks) and in teratomas generated from hESCs. The involvement of vascular smooth muscle cells in the early stages of developing human embryonic blood vessels is demonstrated, as well as the remodeling kinetics of the developing human embryonic blood and lymphatic vasculature. In teratomas, human vascular cells were demonstrated to be associated with developing blood vessels. Processes of intensive remodeling of blood vessels during the early stages of human development are indicated by the upregulation of angiogenic factors and specific structural proteins. At the same time, evidence for lymphatic sprouting and moderate activation of lymphangiogenesis is demonstrated during these developmental stages. In the teratomas induced by hESCs, human angiogenesis and lymphangiogenesis are relatively insignificant. The main source of blood vessels developing within the teratomas is provided by the murine host. We conclude that the teratoma model has only limited value as a model to study human neovasculogenesis and that other in vitro methods for spontaneous and guided differentiation of hESCs may prove more useful.

Advances in human stem cell research.

Daily, scientists join the rapidly growing circle of researchers exploring one of the new frontiers of modern science: stem cells. Just as today medical practice previous to the introduction of antibiotics seems archaic and almost unimaginable, so will the pre“stem cell age” in the not so distant future. The aim of this review is to expose clinicians to the world of stem cells, provide key definitions, and discuss advances in stem cell research and obstacles to be overcome before clinical implementation can take place. Great confusion and many misnomers surround the criteria used to define stem cells. To be considered such, cells must exhibit at least the following characteristics:

Human Vascular Progenitor Cells

Publisher Summary During the process of human embryonic development, blood islands develop alongside the endoderm, which segregate into individual hemangioblasts that are surrounded by flattened endothelial precursor cells. The hemangioblasts mature into the first blood cells, while the endothelial precursors develop into blood vessel endothelium. New vascular formation, termed vasculogenesis, takes place within the embryo. The role of blood cell production is taken over by a series of embryonic organs, such as the liver, spleen, thymus, and bone marrow. The bipotential hemangioblast produces the primitive erythroid and endothelial progenitor cells, and the hemogenic endothelium gives rise to hematopoietic stem cells and endothelial progenitors. This chapter discusses the ability of these progenitors of hematopoiesis (blood cell repopulation), vasculogenesis or angiogenesis. The role of human embryonic stem cells as a source for vascular progenitors is also discussed. Human embryoid bodies (hEBs) are formed by the spontaneous differentiation of hESCs, and comprise multilineage tissues from endodermal, ectodermal, and mesodermal origin. Several experimental procedures have been developed to explore the endothelial potential of hESCs. Undifferentiated human embryonic stem cells (hESCs) form teratomas once injected into severe combined immunodeficient (SCID) mice. During teratoma formation from hESCs, two parallel vascular processes occur: angiogenesis of host vasculature into the forming human teratoma; and vasculogenesis of spontaneously differentiating hESCs. In further studies of human vasculogenesis, various vascular markers should be examined and evaluated in the course of spontaneous differentiation.

30 – Vascular Progenitor Cells in the Human Model

During the third week of human embryo development, blood vessels are formed in conjunction with blood islands within the yolk sac mesoderm. During this process, blood islands develop alongside the endoderm, which segregate into individual hemangioblasts that are surrounded by flattened endothelial precursor cells. The hemangioblasts mature into the first blood cells, whereas the endothelial precursors develop into blood vessel endothelium. At the end of the third week, the entire yolk sac, chorionic villi, and the connecting stalk are vascularized. New vascular formation, termed vasculogenesis, takes place within the embryo on day 18. During this process, the underlying endoderm secretes substances that cause some cells of the splanchnopleuric mesoderm to differentiate into angioblasts. These mesodermal angioblasts then flatten into endothelial cells and coalesce, resulting in small vesicular structures referred to as angiocysts. The ability of these progenitors of hematopoiesis or vasculogenesis, or angiogenesis is yet to be determined. In human embryos, homogenic endothelium could be observed and isolated in both extraembryonic and intraembryonic regions. Culture examination of the hematopoietic potential of embryonic tissue rudiments reveals that hematopoietic potential present inside the embryo include both lymphoid and myeloid lineages, whereas the yolk sac exhibit only myelopoiesis potential, thus questioning its ability to contribute to definitive hematopoiesis in adult human.