Human embryonic stem cell-derived cardiomyocytes (hESC-CMs) provide a promising source for cell therapy and drug screening. Several high-yield protocols exist for hESC-CM production; however, methods to significantly advance hESC-CM maturation are still lacking. Building on our previous experience with mouse ESC-CMs, we investigated the effects of 3-dimensional (3D) tissue-engineered culture environment and cardiomyocyte purity on structural and functional maturation of hESC-CMs. 2D monolayer and 3D fibrin-based cardiac patch cultures were generated using dissociated cells from differentiated Hes2 embryoid bodies containing varying percentage (48-90%) of CD172a (SIRPA)-positive cardiomyocytes. hESC-CMs within the patch were aligned uniformly by locally controlling the direction of passive tension. Compared to hESC-CMs in age (2 weeks) and purity (48-65%) matched 2D monolayers, hESC-CMs in 3D patches exhibited significantly higher conduction velocities (CVs), longer sarcomeres (2.09±0.02 vs. 1.77±0.01 μm), and enhanced expression of genes involved in cardiac contractile function, including cTnT, αMHC, CASQ2 and SERCA2. The CVs in cardiac patches increased with cardiomyocyte purity, reaching 25.1 cm/s in patches constructed with 90% hESC-CMs. Maximum contractile force amplitudes and active stresses of cardiac patches averaged to 3.0±1.1 mN and 11.8±4.5 mN/mm2, respectively. Moreover, contractile force per input cardiomyocyte averaged to 5.7±1.1 nN/cell and showed a negative correlation with hESC-CM purity. Finally, patches exhibited significant positive inotropy with isoproterenol administration (1.7±0.3-fold force increase, EC50 = 95.1 nM). These results demonstrate highly advanced levels of hESC-CM maturation after 2 weeks of 3D cardiac patch culture and carry important implications for future drug development and cell therapy studies.

Hematopoietic development is a complex process that involves a large number of growth factors and cytokines. Many cytokines are known to act on more mature, lineage-restricted cells of the hematopoietic system. However, no specific factors have yet been identified that induce the expansion of the most primitive hematopoietic cells without also inducing differentiation. To search for such factors, we isolated novel cell lines from the yolk sac in order to identify genes important in early hematopoietic and endothelial development. This approach led to the discovery of B219, a sequence that is expressed in at least four isoforms in very primitive hematopoietic cell populations and which may represent a novel hemopoietin receptor. The recently published receptor for the obesity (ob) gene product (leptin) is an isoform of B219 with a nearly identical ligand binding domain. B219/obr is expressed in the yolk sac, early fetal liver, enriched hematopoietic stem cells and in a variety of lymphohematopoietic cell lines. B219/obr is also expressed at high levels in adult reproductive organs. B219/obr maps to human chromosome 1p32, a region syntenic with the recently reported location of obr on murine chromosome 4 (ref. 5).

The expression of leptin and its receptors was examined by reverse transcriptase-polymerase chain reaction and immunofluorescence in granulosa and cumulus cells of pre-ovulatory follicles and in meiotically mature oocytes obtained from women undergoing in-vitro fertilization. Leptin concentrations were measured in newly aspirated follicular fluids and in maternal serum before and after the administration of an ovulatory dose of human chorionic gonadotrophin. The findings demonstrate leptin expression at the mRNA and protein levels by granulosa and cumulus cells, and the presence of leptin in mature human oocytes. While an association between follicular leptin concentration and embryo development was not observed, a post-ovulatory increase in serum leptin concentration was associated with implantation potential. The results are discussed with respect to possible roles of leptin in early human development.

A novel system to study early hematopoietic development is described. This report documents the in vitro capacity of murine embryonic stem (ES) cells to differentiate into hematopoietic precursors of most, if not all, of the colony-forming cells found in normal bone marrow. This system is used to correlate the genetic expression of cytokines, their receptors, the 13-globins, and the hematopoietic cell surface markers throughout the time course of ES cell differentiation with the hematopoietic development that occurs in these cultures. Our results indicate that there is a strong transcriptional activation, in a well-defined temporal order, of most of these genes including erythropoietin (Epo), CSF-1, IL-4, IL-6, I~-globins, as well as the receptors for Epo, CSF-I, and IL-4. IL-3 and GM-CSF were not expressed during the first 24 days of ES cell differentiation. In contrast, the Steel (S/) factor (SLF) was expressed early and underwent substantial up-regulation during this differentiation, and its receptor, c-kit, was expressed relatively constantly throughout the culture period. Our results are consistent with the conclusion that SLF, Epo, IL-4, and IL-6 are important during the early stages of ES cell differentiation and hematopoietic development. Furthermore, these results argue strongly that IL-3 and GM-CSF are not critical to early hematopoiesis. This system offers a unique in vitro model for studying hematopoietic development at the earliest possible stages.

Ly-6A is a murine antigen which is implicated in lymphocyte activation and may be involved in activation of hematopoietic stem cells. Antibody cross-linking studies and antisense experiments have suggested that Ly-6A is a lymphocyte coactivation molecule. To better understand the function of Ly-6A, we used gene targeting to produce Ly-6A null mice which are healthy and have normal numbers and percentages of hematopoietic lineages. However, T lymphocytes from Ly-6A–deficient animals proliferate at a significantly higher rate in response to antigens and mitogens than wild-type littermates. In addition, Ly-6A mutant splenocytes generate more cytotoxic T lymphocytes compared to wild-type splenocytes when cocultured with alloantigen. This enhanced proliferation is not due to alterations in kinetics of response, sensitivity to stimulant concentration, or cytokine production by the T cell population, and is manifest in both in vivo and in vitro T cell responses. Moreover, T cells from Ly-6A–deficient animals exhibit a prolonged proliferative response to antigen stimulation, thereby suggesting that Ly-6A acts to downmodulate lymphocyte responses.

The T-cell antigen receptor is a heterodimeric molecule composed of alpha- and beta-subunits of relative molecular mass 40,000-50,000 (refs 1-6). Recently, the genes encoding both the beta- and alpha- chains have been cloned. By comparing amino-acid and nucleic-acid sequences, it is clear that these genes encode the alpha- and beta-proteins of the T-cell receptor. In addition, a third receptor-like gene, the gamma-chain gene, has been identified, which has many structural and sequence characteristics in common with the alpha- and beta-chain genes. The role of the gamma-chain gene in T-cell development is unknown. We have reported recently that the beta-chain genes are transcriptionally turned on in the thymus at about day 17 of fetal development. Here we report that the alpha-chain also is transcriptionally activated during this time, but that the gamma-chain gene is active in the thymus at day 14, reaches a peak steady-state level at day 15 and rapidly declines thereafter. If the gamma-chain gene has a functional role, it would seem to be involved very early in T-cell development, before the mature T-cell receptor is expressed.

The expression of T-cell antigen receptors during T-cell ontogeny is an important issue that bears directly on such questions as where T-cell tolerance is acquired, at what stage T cells become susceptible to repertoire selection, and why most thymocytes die within the thymus. The thymus rudiment is colonized during days 11 and 12 of gestation, but it is not until day 19 that significant numbers of functional thymocytes are present. Although much is known about the ontogeny of function- and specificity-associated surface molecules such as Ly2 and MT4 (the murine equivalent of human T4) during this period, the ontogeny of the T-cell antigen receptors remains obscure. We have now addressed this question on three levels: DNA rearrangement, messenger RNA transcription and expression of cell-surface receptor-like proteins. Our results suggest that T-cell receptors are first expressed within the thymus around day 17 of gestation, independently of and probably before the expression of Ly2 and MT4. Furthermore, these data suggest that all major adult thymocyte subpopulations, including the small cortical cells, most of which die within the thymus, express receptors.

Purpose: The Mer receptor tyrosine kinase, cloned from a B-lymphoblastoid library, is the mammalian orthologue of the chicken retroviral oncogene v-eyk and sends antiapoptotic and transforming signals when activated. To determine if Mer expression is ectopic in T-cell acute lymphoblastic leukemia (ALL) and potentially important in leukemogenesis, we analyzed Mer expression in normal human thymocytes and lymphocytes and in pediatric ALL patient samples. Experimental Design: Reverse transcription-PCR, flow cytometry, and immunohistochemistry were used to determine expression of Mer in sorted human thymocyte populations, lymphocytes, and lymphocytes activated by phytohemagglutinin or phorbol 12-myristate 13-acetate/ ionophore. Mer expression in 34 T-cell ALL (T-ALL) patient samples was evaluated by reverse transcription-PCR, and Mer protein expression in a separate cohort of 16 patient samples was assayed by flow cytometry and Western blot. Results: Mer expression was absent in normal thymocytes or lymphocytes, and in T cells activated with phytohemagglutinin or phorbol 12-myristate 13-acetate/ionophore. In contrast, Jurkat cells and T-ALL patient samples expressed unique 180 to 185 kDa Mer protein glycoforms. Substantial Mer RNA levels were principally observed in a subset of T-ALL patient samples that expressed B220 (P = 0.004) but lacked surface expression of CD3 (P = 0.02) and CD4 (P = 0.006), a phenotypic profile consistent with immature lymphoblasts. In addition, 8 of 16 T-ALL patient samples had Mer protein detected by flow cytometry and Western blot. Conclusions: Transforming Mer signals may contribute to T-cell leukemogenesis, and abnormal Mer expression may be a novel therapeutic target in pediatric ALL therapy.Acute lymphoblastic leukemia (ALL) is the most common childhood malignancy. The current 80% 5-year event-free survival rate is a product of national pediatric oncology cooperative group clinical trials that have optimized patient outcome. Future improvements will rely on a better understanding of the definition and pathogenesis of different ALL subtypes. Specifically, biologically targeted therapy against unique leukemia markers or signaling pathways is being tested in clinical trials and hopefully will complement current treatment regimens. In this study, we report a new potential biological marker in pediatric ALL.Mer was initially cloned by our group from a human B lymphoblastoid cDNA library, and Mer RNA transcript was also found to be expressed in T-cell ALL (T-ALL) cell lines (1). This was intriguing because Mer RNA was not detected in freshly isolated human (or mouse) T or B lymphocytes. Subsequent work has shown that Mer signaling, via activated Mer receptor tyrosine kinase domain, can be antiapoptotic (32D cells; ref.2), cytoskeletal regulatory, or frankly transforming [Ba/F3 pro-Blymphocytes (3) and NIH 3T3 fibroblasts (4)], depending on the cell type. The retrovirally transduced chicken orthologue v-eyk was also frankly transforming (5, 6). We now show that Mer expression is abs...