Biomedical subjects

Joseph Itskovitz-Eldor

Publications and source records attributed to Joseph Itskovitz-Eldor.

At least 37 records · Page 2Linked to original sources

PubMed2005

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 (alpha-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.

Biomarkers↗

PubMed2005

Functional properties of human embryonic stem cell-derived cardiomyocytes.

Regeneration of the diseased myocardium by cardiac cell transplantation is an attractive therapeutic modality. Yet, because the transplanted cardiomyocytes should functionally integrate within the diseased myocardium, it is preferable that their properties resemble those of the host. To determine the functional adaptability of human embryonic stem cell-derived cardiomyocytes (hESC-CM) to the host myocardium, the authors investigated the excitation-contraction (E-C) coupling and the responsiveness to common physiological stimuli. The main findings are: (1) hESC-CM readily respond to electrical pacing and generate corresponding [Ca(2+)](i) transients (measured by fura-2 fluorescence) and contractions (measured by video edge detector). (2) In contrast to the mature myocardium, hESC-CM display negative force-frequency relations. (3) The hESC-CM contraction is dependent on [Ca(2+)](o) and blocked by verapamil. (4) Surprisingly, ryanodine, the sarcoplasmic-endoplasmic reticulum Ca(2+)-ATPase inhibitor thapsigargin, and caffeine do not affect the [Ca(2+)](i) transient or contraction. Collectively, these results indicate that at the developmental stage of 45 to 60 days, the contraction is largely dependent on [Ca(2+)](o) rather than on sarcoplasmic reticulum (SR) Ca(2+) stores. The results show for the first time that the E-C coupling properties of hESC-CM differ from the adult myocardium, probably due to immature SR function. Based on these findings, genetic manipulation of hESC-CM toward the adult myocardium should be considered.

Animals↗

PubMed2005

High-efficiency RNA interference in human embryonic stem cells.

RNA interference methodology suppresses gene expression, thus mimicking loss-of-function mutation and enabling in vitro and in vivo gene function analysis. In this study, we used retroviral and lentiviral vectors to deliver small interfering RNAs and report high-efficiency silencing of a green fluorescent protein (GFP) trans gene and the stem cell-specific transcription factors Oct4/POU5F1 and Nanog in human embryonic stem cells. Gene knockdown of Oct4 and Nanog promotes differentiation, thereby demonstrating a role for these factors in human embryonic stem cell self-renewal.

Antigens, Surface↗

PubMed2005

CD133-positive hematopoietic stem cell "stemness" genes contain many genes mutated or abnormally expressed in leukemia.

Affymetrix human Hu133A oligonucleotide arrays were used to study the expression profile of CD133+ cord blood (CB) and peripheral blood (PB) using CD133 cell-surface marker. An unsupervised hierarchical clustering of 14,025 valid probe sets showed a clear distinction between the CD133+ cells representing the hematopoietic stem cell (HSC) population and CD133-differentiated cells. Two hundred forty-four genes were found to be upregulated by at least twofold in the CD133-positive cells of both CB and PB compared with the CD133-negative cells. These genes represent the hematopoietic "stemness," whereas the 218 and 304 upregulated genes exclusively in PB and CB, respectively, represent tissue specificity. Some of the stemness genes were also common to HSC genes found to be upregulated in several recently published studies. Among these common stemness genes, we identified several groups of genes that have an important role in hematopoiesis: growth factor receptors, transcription factors, genes that have an important role in development, and genes involved in cell growth. Sixteen selected stemness genes are known to be mutated or abnormally regulated in acute leukemias. It can be suggested that key hematopoietic stemness machinery genes may lead to abnormal proliferation and leukemia upon mutation or change of their expression.

AC133 Antigen↗

PubMed2005

[Insemination from HIV-positive males].

INTRODUCTION: Discordant couples, in which the man is HIV positive and the woman is HIV negative, face limited options when they wish to produce healthy children whilst practicing safe sex. In order to conceive they must abandon unprotected sex, which carries high risk of HIV infection to the woman. OBJECTIVE: To develop a method for removing the HIV from semen and then perform intra uterine insemination, following verification of the HIV-negative spermatozoa fraction by PCR. METHODS: Motile spermatozoa were isolated from semen samples by the gradient and "swim-up" techniques. HIV-RNA was tested before and after the procedure in both the semen and the purified spermatozoa fraction. Insemination was performed on ovulation day only when the absence of any viral particles was verified by PCR. RESULTS: Four couples underwent a total of 8 cycles of intra uterine insemination (IUI). Presently, two healthy babies were born. The two mothers and newborn were found to be HIV negative following the delivery. CONCLUSION: We report a safe procedure which enables biological parenthood for HIV-1 discordant couples without the risk of infecting the female partner. Our clinical data corroborate with the collaborative European data on this subject.

Adult↗

PubMed2005

[Episiotomy as a risk factor for early perineal trauma].

OBJECTIVE: To assess the association between medio-lateral episiotomy and early perineal trauma. STUDY DESIGN: A prospective observational cohort study of 667 consecutive vaginal deliveries was conducted over a 10-week period. On the day following delivery, all women were examined for the extent of early perineal trauma (swelling, oedema, haematoma), in addition to the episiotomy or lacerations. The demographic and clinical variables (age, parity, gestational age, birthweight, length of second stage, operative delivery, epidural, seniority of the obstetrical provider, episiotomy) and the post-partum treatment given for the perineum were retrieved from the hospital records. RESULTS: The overall episiotomy rate was 37.6%. Significant early perineal trauma was found in 9.9% of the women, more so in the episiotomy group (17.1% vs 5.5%, p < 0.001). In the episiotomy group, a larger number of women received more than routine treatment for the perineum (6.4% vs 2.9%, p < 0.05). Episiotomy was found to be the major independent predictor for perineal trauma (OR: 4.5, 95% CI: 2.4-8.5, p < 0.001), followed by epidural analgesia (OR: 2.4, CI: 1.3-4.3, p < 0.05) and birth weight >3700 g (OR: 2.1, CI: 1.1-4.1, p < 0.05). CONCLUSION: Episiotomy does not prevent immediate postpartum perineal trauma; rather, it is a major independent risk factor associated with significant early perineal trauma.

Adult↗

PubMed2004

Three-dimensional porous alginate scaffolds provide a conducive environment for generation of well-vascularized embryoid bodies from human embryonic stem cells.

Differentiation of human embryonic stem cells (hESCs) can be instigated through the formation of embryo-like aggregates in suspension, termed human embryoid bodies (hEBs). Controlling cell aggregation and agglomeration during hEBs formation has a profound effect on the extent of cell proliferation and differentiation. In a previous work, we showed that control over hEBs formation and differentiation can be achieved via cultivation of hESC suspensions in a rotating bioreactor system. We now report that hEBs can be generated directly from hESC suspensions within three-dimensional (3D) porous alginate scaffolds. The confining environments of the alginate scaffold pores enabled efficient formation of hEBs with a relatively high degree of cell proliferation and differentiation; encouraged round, small-sized hEBs; and induced vasculogenesis in the forming hEBs to a greater extent than in static or rotating cultures. We therefore conclude that differentiation of hEBs can be induced and directed by physical constraints in addition to chemical cues.

Alginates↗

PubMed2004

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.

Cell Differentiation↗

PubMed2004

Electromechanical integration of cardiomyocytes derived from human embryonic stem cells.

Cell therapy is emerging as a promising strategy for myocardial repair. This approach is hampered, however, by the lack of sources for human cardiac tissue and by the absence of direct evidence for functional integration of donor cells into host tissues. Here we investigate whether cells derived from human embryonic stem (hES) cells can restore myocardial electromechanical properties. Cardiomyocyte cell grafts were generated from hES cells in vitro using the embryoid body differentiating system. This tissue formed structural and electromechanical connections with cultured rat cardiomyocytes. In vivo integration was shown in a large-animal model of slow heart rate. The transplanted hES cell-derived cardiomyocytes paced the hearts of swine with complete atrioventricular block, as assessed by detailed three-dimensional electrophysiological mapping and histopathological examination. These results demonstrate the potential of hES-cell cardiomyocytes to act as a rate-responsive biological pacemaker and for future myocardial regeneration strategies.

Animals↗

PubMed2004

Vascular development in early human embryos and in teratomas derived from human embryonic stem cells.

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.

Animals↗

PubMed2004

Bioreactor cultivation enhances the efficiency of human embryoid body (hEB) formation and differentiation.

The promise of human embryonic stem cells (hESCs) to provide an unlimited supply of cells for cell therapy and tissue engineering depends on the availability of a controllable bioprocess for their expansion and differentiation. We describe for the first time the formation of differentiating human embryoid bodies (hEBs) in rotating bioreactors to try and control their agglomeration. The efficacy of the dynamic process compared to static cultivation in Petri dishes was analyzed with respect to the yield of hEB formation and differentiation. Quantitative analyses of hEBs, DNA and protein contents, and viable cell concentration, as measures for culture cellularity and scale-up, revealed 3-fold enhancement in generation of hEBs compared to the static culture. Other metabolic indices such as glucose consumption, lactic acid production, and pH pointed to efficient cell expansion and differentiation in the dynamic cultures. The type of rotating vessel had a significant impact on the process of hEB formation and agglomeration. In the slow turning lateral vessel (STLV), hEBs were smaller in size and no large necrotic centers were seen, even after 1-month cultivation. In the high aspect rotating vessel (HARV), hEB agglomeration was massive. The appearance of representative tissues derived from the three germ layers as well as primitive neuronal tube organization, blood vessel formation, and specific-endocrine secretion indicated that the initial developmental events are not altered in the dynamically formed hEBs. Collectively, our study defines the culture conditions in which control over the aggregation of differentiating hESCs is obtained, thus enabling scaleable cell production for clinical and industrial applications.

Apoptosis↗

PubMed2004

Differences between human and mouse embryonic stem cells.

We compared gene expression profiles of mouse and human ES cells by immunocytochemistry, RT-PCR, and membrane-based focused cDNA array analysis. Several markers that in concert could distinguish undifferentiated ES cells from their differentiated progeny were identified. These included known markers such as SSEA antigens, OCT3/4, SOX-2, REX-1 and TERT, as well as additional markers such as UTF-1, TRF1, TRF2, connexin43, and connexin45, FGFR-4, ABCG-2, and Glut-1. A set of negative markers that confirm the absence of differentiation was also developed. These include genes characteristic of trophoectoderm, markers of germ layers, and of more specialized progenitor cells. While the expression of many of the markers was similar in mouse and human cells, significant differences were found in the expression of vimentin, beta-III tubulin, alpha-fetoprotein, eomesodermin, HEB, ARNT, and FoxD3 as well as in the expression of the LIF receptor complex LIFR/IL6ST (gp130). Profound differences in cell cycle regulation, control of apoptosis, and cytokine expression were uncovered using focused microarrays. The profile of gene expression observed in H1 cells was similar to that of two other human ES cell lines tested (line I-6 and clonal line-H9.2) and to feeder-free subclones of H1, H7, and H9, indicating that the observed differences between human and mouse ES cells were species-specific rather than arising from differences in culture conditions.

Animals↗

PubMed2004

Cardiovascular potential of embryonic stem cells.

Initial events involved in the process of heart formation consist of myocardial differentiation as well as development of endothelial and endocardial tissues. As only limited means are allocated to the studying of cardiovascular system development, embryonic stem cells (ESCs) isolated from the inner cell mass (ICM) of developing mice or human blastocysts offer the first step toward the understanding of these complex and intriguing events. ESCs are able to differentiate into a wide range of cell types, including various vascular cells and cardiomyocytes, and their self-renewal capability renders them a unique, homogeneous, and unlimited preliminary population of cells for the investigation of early developmental events of cardiovascular system and lineage commitment. This review summarizes the accumulated knowledge of the cellular and molecular mechanisms involved in the development of the cardiovascular system.

Animals↗

PubMed2004

The promise of human embryonic stem cells.

Pluripotency refers to the ability of a cell to give rise to cells that originate from all three germ layers. Among the available human pluripotent cells, human embryonic stem cells (hESCs) are considered to have the greatest probability for practical clinical application because of their simple propagation and stability in culture. Since their first derivation, issues concerning hESC maintenance and self-renewal have been widely addressed. The first part of this review presents the accumulated knowledge concerning the self-renewal of hESCs and discusses recent genetic profile data, which seem to shed light on hESC self-renewal and pluripotency mechanism. The second part deals with the regenerative potential of hESCs. Available lineage-specific differentiations of hESCs are presented, with detailed data on the ability of hESCs to differentiate into trophoblast cells, an observation that might broaden the definition of their developmental potential. Specific focus is given to vascular cell differentiation, including endothelial and smooth muscle cells. Transplantation limitations as well as current steps taken toward resolution conclude the review.

Blastocyst↗

PubMed2004

Cell therapy using human embryonic stem cells.

Cell therapy refers to the transplantation of healthy, functional and propagating cells to restore the viability or function of deficient tissues. Stem cells are characterized by self-renewal and the potential to form differentiated cells. In early mammalian embryos, at the blastocyst stage, the inner cell mass is pluripotent. Thus, it has been recognized that human embryonic stem cells (hESCs), which are derived from such cells of blastocysts, may serve as a source of numerous types of differentiated cells. The first part of this review summarizes different techniques for the derivation and maintenance of undifferentiated hESCs. In the second part, issues concerning the safety and bulk production, which may enable hESCs use in future clinical applications, are presented. The last part of this review details accumulated data regarding the in vitro differentiation potential of hESCs.

Blastocyst↗

PubMed2004

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, Parkinson's 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.

Cardiomyopathies↗

PubMed2004

Developing the role of the social worker as coordinator of services at the surrogate parenting center.

A law permitting couples to conceive biological children through surrogacy was legislated in Israel in March 1996. The Rambam Medical Center has established the only nonprofit Surrogate Parenting Center at a public hospital in Israel. The multidisciplinary teamwork at the Center is case managed by a social worker. An important role of the social work intervention is consultation and support for the couple and the surrogate at all stages of the process. The case study presented in the article illustrates the need for sensitive and professional intervention due to the complexity of the surrogacy process and the crisis it involves for both the surrogate and the couple. In light of the growing parenting surrogacy cases in the United States, Europe, and Israel, a structured social work intervention model is described, which may be implemented at public or private surrogate parenting centers.

Adaptation, Psychological↗

PubMed2004

Differentiation of human embryonic stem cells into insulin-producing clusters.

Type I diabetes mellitus is caused by an autoimmune destruction of the insulin-producing beta 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 beta cells for transplantation. We present here a method for forming immature islet-like clusters of insulin-producing cells derived from hES cells. The protocol consisted of several steps. Embryoid bodies were first cultured and plated in insulin-transferrin-selenium-fibronectin medium, followed by medium supplemented with N2, B27, and basic fibroblast growth factor (bFGF). Next, the glucose concentration in the medium was lowered, bFGF was withdrawn, and nicotinamide was added. Dissociating the cells and growing them in suspension resulted in the formation of clusters which exhibited higher insulin secretion and had longer durability than cells grown as monolayers. Reverse transcription-polymerase chain reaction detected an enhanced expression of pancreatic genes in the differentiated cells. Immunofluorescence and in situ hybridization analyses revealed a high percentage of insulin-expressing cells in the clusters. In addition to insulin, most cells also coexpressed glucagon or somatostatin, indicating a similarity to immature pancreatic cells. Further improvement of this insulin-producing cell protocol may lead to the formation of an unlimited source of cells suitable for transplantation.

Apoptosis↗