Multipotent hematopoietic progenitor cells immortalized by Lhx2 self-renew by a cell nonautonomous mechanism

OBJECTIVE: Direct molecular and cellular studies of hematopoietic stem cells (HSCs) are hampered by the low levels of HSCs in hematopoietic tissues. To address these issues, we generated immortalized multipotent hematopoietic precursor cell (HPC) lines by expressing the LIM-homeobox gene Lhx2 (previously LH2) in hematopoietic progenitors derived from embryonic stem cells differentiated in vitro. MATERIALS AND METHODS: To validate further the relevance of the HPC lines as a model for normal HSCs, we analyzed in detail the growth requirements of HPC lines in vitro. RESULTS: Lhx2 immortalized the HPC lines by a putatively novel and cell nonautonomous mechanism. Self-renewal of the HPC lines is dependent on functional Lhx2 expression. Most early-acting hematopoiesis-related growth factors show synergistic effects on the HPC lines, whereas late-acting factors do not induce differentiation by themselves. Transforming growth factor-beta(1) is a potent inhibitor of proliferation of the HPC lines. HPC lines form cobblestone areas with high efficiency when seeded onto stromal cell lines, and the cobblestone area-forming cell can be maintained in these cultures for several months. CONCLUSIONS: Our data show that, in many respects, HPC lines are similar to normal hematopoietic progenitor/stem cells on the cellular level, in contrast to most previously described multipotent hematopoietic cell lines. The cell nonautonomous mechanism for immortalization of the HPC lines suggests that Lhx2 regulates, directly or indirectly, soluble mediators involved in self-renewal of the HPC lines.     

Hematopoietic chimerism in liver transplantation patients and hematopoietic stem/progenitor cells in adult human liver

Liver transplantation (LT) is a cure for many liver diseases. Blood chimerism of donor origin can develop after LT, which raises the possibility of the existence of hematopoietic stem/progenitor cells (HSPCs) in the liver. We characterized the blood chimerism in a large cohort of 249 LT patients and analyzed putative HSPCs in adult human livers. The overall incidence of chimerism was 6.43%, of which 11.11% was among short-term (1 day to 6 months) and 3.77% was among long-term (6 months to 8 years) LT patients. Hematopoietic Lin(-) CD34(+) CD38(-) CD90(+) populations have been demonstrated to generate long-term lymphomyeloid grafts in transplantations. In human adult livers, we detected Lin(-) CD34(+) CD38(-) CD90(+) populations accounting for 0.03% ± 0.017% of the total single liver cells and for 0.05% ± 0.012% of CD45(+) liver cells. Both Lin(-) CD34(+) and Lin(-) CD45(+) liver cells, from extensively perfused human liver grafts, were capable of forming hematopoietic myeloid-lineage and erythroid-lineage methylcellulose colonies. More importantly, Lin(-) CD45(+) or CD45(+) liver cells could be engrafted into hematopoietic cells in an immunodeficient mouse model. These results are the first evidence of the presence of putative HSPC populations in the adult human liver, where the liver is a good ectopic niche. The discovery of the existence of HSPCs in the adult liver have implications for the understanding of extramarrow hematopoiesis, liver regeneration, mechanisms of tolerance in organ transplantation, and de novo cancer recurrence in LT patients. Conclusion: The human adult liver contains a small population of HSPCs. In LT patients, there are two types of chimerisms: transient chimerism, resulting from mature leucocytes, and long-term chimerism, derived from putative HSPCs in the liver graft. (HEPATOLOGY 2012).     

Ex vivo expansion of human hematopoietic stem and progenitor cells

Despite progress in our understanding of the growth factors that support the progressive maturation of the various cell lineages of the hematopoietic system, less is known about factors that govern the self-renewal of hematopoietic stem and progenitor cells (HSPCs), and our ability to expand human HSPC numbers ex vivo remains limited. Interest in stem cell expansion has been heightened by the increasing importance of HSCs in the treatment of both malignant and nonmalignant diseases, as well as their use in gene therapy. To date, most attempts to ex vivo expand HSPCs have used hematopoietic growth factors but have not achieved clinically relevant effects. More recent approaches, including our studies in which activation of the Notch signaling pathway has enabled a clinically relevant ex vivo expansion of HSPCs, have led to renewed interest in this arena. Here we briefly review early attempts at ex vivo expansion by cytokine stimulation followed by an examination of our studies investigating the role of Notch signaling in HSPC self-renewal. We will also review other recently developed approaches for ex vivo expansion, primarily focused on the more extensively studied cord blood-derived stem cell. Finally, we discuss some of the challenges still facing this field.     

Thrombopoietin stimulation of hematopoietic stem/progenitor cells

The recent cloning of the thrombopoietin gene, and the production of recombinant protein, have allowed studies on both its biological actions and clinical utility. Thrombopoietin not only affects the cells of the megakaryocytic lineage, but has a diverse set of cellular targets. In particular, it stimulates the ex vivo expansion of hematopoietic stem/progenitor cells suggesting that it may play a role in transplantation studies. Pre-clinical but limited clinical studies indicate that under defined conditions, thrombopoietin may accelerate white blood cell count and platelet recoveries following myelosuppression or radiotherapy.     

Ex vivo expansion of hematopoietic stem and progenitor cells: Recent advances

Hematopoietic stem cells(HSCs) have become the most extensively studied stem cells and HSC-based cellular therapy is promising for hematopoietic cancers and hereditary blood disorders. Successful treatment of patients with HSC cells depends on sufficient number of highly purified HSCs and progenitor cells. However, stem cells are a very rare population no matter where they come from. Thus, ex vivo amplification of these HSCs is essential. The heavy demands from more and more patients for HSCs also require industrial-scale expansion of HSCs with lower production cost and higher efficiency. Two main ways to reach that goal:(1) to find clinically applicable, simple and efficient methods(or reagents) to enrich HSCs;(2) to find new developmental regulators and chemical compounds in order to replace the currently used cytokine cocktails for HSCsamplification. In this Editorial review, we would like to introduce the current status of ex vivo expansion of HSCs, particularly focusing on enrichment and culture supplements.     

Hematopoietic stem cells and other hematopoietic cells show broad resistance to chemotherapeutic agents in vivo when overexpressing bcl-2

Objective. Chemotherapeutic agents function by inducing apoptosis and their effectiveness depends on the balance of pro- and anti-apoptotic proteins in cells. Due to the complicated interactions of the many proteins involved, it has been difficult to determine in tumors whether overexpression of single genes is prognostic for increased resistance. Therefore, we studied the influence of bcl-2 overexpression on resistance to chemotherapeutics in a transgenic mouse system. This allowed us to study a wide variety of cells, including important but rare populations such as hematopoietic stem cells (HSC).Methods. H2K-bcl-2 transgenic and wild-type (WT) mice were treated with several agents(5-fluoruracil, cyclophosphamide, and busulfan) to determine the contribution of increased amounts of bcl-2 to the response to these chemotherapeutics in vivo. Populations were enumerated using flow cytometry. HSC were studied by FACS purification and long-term reconstitution assays in vivo and resistance was confirmed by short-term proliferation assays with different amounts of chemotherapeutics in vitro.Results. bcl-2 overexpression alone protects many cell types, though protection levels differ between populations and agents. However, even sensitive populations return to pretreatment levels faster in transgenic mice. bcl-2 overexpression also prevents the dramatic changes in HSC following 5-FU treatment (downregulation of c-kit, upregulation of Lin, less efficient long-term reconstitution). In vitro studies directly demonstrate increased resistance of bcl-2 overexpressing HSC to chemotherapeutic agents.Conclusions. Increased expression of bcl-2 in HSC and their progeny endows these cells with broad resistance to chemotherapeutic agents. The ability to (differentially) regulate sensitivity to apoptosis of bystander and tumor cells is clinically important.     

Single Lgr5- or Lgr6-expressing taste stem/progenitor cells generate taste bud cells ex vivo

Leucine-rich repeat-containing G protein-coupled receptor 5 (Lgr5) and its homologs (e.g., Lgr6) mark adult stem cells in multiple tissues. Recently, we and others have shown that Lgr5 marks adult taste stem/progenitor cells in posterior tongue. However, the regenerative potential of Lgr5-expressing (Lgr5⁺) cells and the identity of adult taste stem/progenitor cells that regenerate taste tissue in anterior tongue remain elusive. In the present work, we describe a culture system in which single isolated Lgr5⁺ or Lgr6⁺ cells from taste tissue can generate continuously expanding 3D structures (“organoids”). Many cells within these taste organoids were cycling and positive for proliferative cell markers, cytokeratin K5 and Sox2, and incorporated 5-bromo-2’-deoxyuridine. Importantly, mature taste receptor cells that express gustducin, carbonic anhydrase 4, taste receptor type 1 member 3, nucleoside triphosphate diphosphohydrolase-2, or cytokeratin K8 were present in the taste organoids. Using calcium imaging assays, we found that cells grown out from taste organoids derived from isolated Lgr5⁺ cells were functional and responded to tastants in a dose-dependent manner. Genetic lineage tracing showed that Lgr6⁺ cells gave rise to taste bud cells in taste papillae in both anterior and posterior tongue. RT-PCR data demonstrated that Lgr5 and Lgr6 may mark the same subset of taste stem/progenitor cells both anteriorly and posteriorly. Together, our data demonstrate that functional taste cells can be generated ex vivo from single Lgr5⁺ or Lgr6⁺ cells, validating the use of this model for the study of taste cell generation.Taste bud cells are heterogeneous and undergo constant turnover (1); however, the origins and generation of taste buds in adult mammals remain largely unclear. Based on morphological and functional characteristics, there are at least three different types of mature taste bud cells [type 1 (glial-like cells), type 2 (receptor cells, including those responsible for sensing sweet, bitter, and umami stimuli), and type 3 (presynaptic cells, including sour sensors)], and well as one type of immature taste bud cell [type 4 (basal cells that are precursors of other types of mature taste cells)] (2, 3). Mature taste bud cells are postmitotic and short-lived, with average life spans estimated at 8–12 d (4, 5), although distinct subtypes of taste bud cells may have different life spans (1, 4, 5). At present, the stem cell population and the regenerative process from adult taste stem/progenitor cells to mature taste bud cells are not well characterized.Lgr5 (leucine-rich repeat-containing G protein-coupled receptor 5), encoded by a Wnt (wingless-type MMTV integration site family) target gene, marks adult stem/progenitor cells in taste tissue in posterior tongue that in vivo give rise to all major types of taste bud cells, as well as perigemmal cells (6, 7). Lgr5 is also known to mark actively cycling stem cells in small intestine, colon, stomach, and hair follicle, as well as quiescent stem cells in liver, pancreas, and cochlea (8). Isolated Lgr5⁺ adult stem cells from multiple tissues are able to generate so-called organoid structures ex vivo (9–11). For instance, Sato and colleagues (10) developed a 3D culture system to grow crypt-villus organoids from single intestinal stem cells; all differentiated cell types were found in these structures, indicating the multipotent nature of these cells. We hypothesized that Lgr5⁺ taste stem/progenitor cells in a 3D culture system would be capable of expanding and giving rise to taste receptor cells ex vivo. In the present study, we isolated Lgr5⁺ stem/progenitor cells from taste tissue and cultured them in a 3D culture system. Single Lgr5⁺ cells grew into organoid structures ex vivo in defined culture conditions, with the presence of both proliferating cells and differentiated mature taste cells in which taste signaling components are functionally expressed. When organoids were replated onto a 2D surface precoated with laminin and polylysine, cells grew out of the organoids and attached to the flat surface, and some cells retained the expressed taste signaling elements and responded to taste stimuli.Lgr5 marks adult taste stem/progenitor cells in posterior tongue, which was shown using an engineered mouse model in which enhanced green fluorescent protein (EGFP) and tamoxifen-inducible Cre recombinase (CreERT2) are knocked-in to replace the coding sequence of Lgr5 and act as surrogate markers for Lgr5 (6, 7). Although Lgr5 is present in fungiform papillae in anterior tongue during embryonic stages and early life, based on the intrinsic GFP signal from the Lgr5-EGFP transgene, Lgr5-EGFP signal could not be detected in fungiform papillae cells in adult mice (6, 7). Therefore, taste stem/progenitor cells remain to be identified in fungiform papillae in anterior tongue. We hypothesized that Lgr6, an Lgr5 homolog, may mark adult taste stem/progenitor cells in anterior tongue, prompted by the finding that Lgr6 is preferentially expressed in taste tissue, but not in the surrounding epithelium devoid of taste tissue (12). Using the Lgr6-EGFP-ires-CreERT2 mouse line (13), we here show that Lgr6 is expressed in cells at the basal area of taste buds in fungiform and circumvallate papillae. By genetic lineage tracing, we show that Lgr6⁺ cells give rise to taste bud cells in taste papillae in both anterior and posterior tongue. RT-PCR shows that Lgr5 and Lgr6 may mark the same subset of taste stem/progenitor cells both anteriorly and posteriorly. Similar to Lgr5⁺ cells, isolated Lgr6⁺ cells can build taste organoids that generate mature taste cells.     

Hematopoietic stem cells and the aging hematopoietic system

The etiology of the age-associated pathophysiological changes of the hematopoietic system including the onset of anemia, diminished adaptive immune competence, and myelogenous disease development are underwritten by the loss of normal homeostatic control. As tissue and organ homeostasis in adults is primarily mediated by the activity of stem and progenitor cells, it has been suggested that the imbalances accompanying aging of the hematopoietic system may stem from alterations in the prevalence and/or functional capacity of hematopoietic stem cells (HSCs) and progenitors. In this review, we examine evidence implicating a role for stem cells in the aging of the hematopoietic system, and focus on the mechanisms suggested to contribute to stem cell aging.     

Redefining endothelial progenitor cells via clonal analysis and hematopoietic stem/progenitor cell principals

The limited vessel-forming capacity of infused endothelial progenitor cells (EPCs) into patients with cardiovascular dysfunction may be related to a misunderstanding of the biologic potential of the cells. EPCs are generally identified by cell surface antigen expression or counting in a commercially available kit that identifies "endothelial cell colony-forming units" (CFU-ECs). However, the origin, proliferative potential, and differentiation capacity of CFU-ECs is controversial. In contrast, other EPCs with blood vessel-forming ability, termed endothelial colony-forming cells (ECFCs), have been isolated from human peripheral blood. We compared the function of CFU-ECs and ECFCs and determined that CFU-ECs are derived from the hematopoietic system using progenitor assays, and analysis of donor cells from polycythemia vera patients harboring a Janus kinase 2 V617F mutation in hematopoietic stem cell clones. Further, CFU-ECs possess myeloid progenitor cell activity, differentiate into phagocytic macrophages, and fail to form perfused vessels in vivo. In contrast, ECFCs are clonally distinct from CFU-ECs, display robust proliferative potential, and form perfused vessels in vivo. Thus, these studies establish that CFU-ECs are not EPCs and the role of these cells in angiogenesis must be re-examined prior to further clinical trials, whereas ECFCs may serve as a potential therapy for vascular regeneration.     

Long-term maintenance of human hematopoietic stem/progenitor cells by expression of BMI1

The polycomb group (PcG) gene BMI1 has been identified as one of the key epigenetic regulators of cell fates during different stages of development in multiple murine tissues. In a clinically relevant model, we demonstrate that enforced expression of BMI1 in cord blood CD34(+) cells results in long-term maintenance and self-renewal of human hematopoietic stem and progenitor cells. Long-term culture-initiating cell frequencies were increased upon stable expression of BMI1 and these cells engrafted more efficiently in NOD-SCID mice. Week 5 cobblestone area-forming cells (CAFCs) were replated to give rise to secondary CAFCs. Serial transplantation studies in NOD-SCID mice revealed that secondary engraftment was only achieved with cells overexpressing BMI1. Importantly, BMI1-transduced cells proliferated in stroma-free cytokine-dependent cultures for more than 20 weeks, while a stable population of approximately 1% to 5% of CD34(+) cells was preserved that retained colony-forming capacity. Whereas control cells lost most of their NOD-SCID engraftment potential after 10 days of ex vivo culturing in absence of stroma, NOD-SCID multilineage engraftment was retained by overexpression of BMI1. Thus, our data indicate that self-renewal of human hematopoietic stem cells is enhanced by BMI1, and we classify BMI1 as an intrinsic regulator of human stem/progenitor cell self-renewal.     

Migratory path of definitive hematopoietic stem/progenitor cells during zebrafish development

The development of vertebrate definitive hematopoiesis is featured by temporally and spatially dynamic distribution of hematopoietic stem/progenitor cells (HSPCs). It is proposed that the migration of definitive HSPCs, at least in part, accounts for this unique characteristic; however, compelling in vivo lineage evidence is still lacking. Here we present an in vivo analysis to delineate the migration route of definitive HSPCs in the early zebrafish embryo. Cell-marking analysis was able to first map definitive HSPCs to the ventral wall of dorsal aorta (DA). These cells were subsequently found to migrate to a previously unappreciated organ, posterior blood island (PBI), located between the caudal artery and caudal vein, and finally populate the kidney, the adult hematopoietic organ. These findings demonstrate that the PBI acts as an intermediate hematopoietic organ in a manner analogous to the mammalian fetal liver to sustain definitive hematopoiesis before adult kidney hematopoiesis occurs. Thus our study unambiguously documents the in vivo trafficking of definitive HSPCs among developmentally successive hematopoietic compartments and underscores the ontogenic conservation of definitive hematopoiesis between zebrafish and mammals.     

Stromal cell CD9 regulates differentiation of hematopoietic stem/progenitor cells

CD9 belongs to the transmembrane 4 superfamily, and has been shown to influence cell proliferation, motility, and adhesion. We show here that ligation of CD9 modifies proliferation and/or differentiation of hematopoietic stem/progenitors. Pluripotent EML-C1 hematopoietic cells were cocultured with MS-5 stromal cells in the presence of KMC8.8, an anti-CD9 antibody. Numbers of recovered EML-C1 cells were slightly reduced and the antibody caused the hematopoietic cells to migrate beneath the adherent stromal cell layer. Of particular interest, EML-C1 cells recovered from CD9-ligated cultures had undifferentiated properties. Separate pretreatment of the two cell types with antibody showed that stromal-cell CD9 mediated these responses. Spontaneous expression of erythroid marker was completely blocked and there was a shift towards undifferentiated clonogenic progenitors. Immunoprecipitation studies showed that stromal-cell CD9 associates with the beta1 subunit of integrin, as well as a novel 100 kD protein. Antibody cross-linking of cell surface CD9 increased the amount of 100 kD protein that was subsequently coprecipitated with CD9. These observations show that stromal-cell CD9 influences physical interactions with hematopoietic cells and may be one factor that determines the degree of stem cell differentiation.     

Ex vivo expansion of hematopoietic progenitor cells and mature cells

Hematopoietic cells have the potential for providing benefit in a variety of clinical settings. These include cells for support of patients undergoing high-dose chemotherapy, as a target for replacement gene therapy, and as a source of cells for immunotherapy. The limitation to many of these applications has been the total absolute number of defined target cells. Therefore many investigators have explored methods to culture hematopoietic cells in vitro to increase the numbers of these cells. Studies attempting to expand hematopoietic stem cells, progenitor cells, and mature cells in vitro have become possible over the past decade due to the availability of recombinant growth factors and cell selection technologies. To date, no studies have demonstrated convincing data on the expansion of true stem cells, and so the focus of this review is the expansion of committed progenitor cells and mature cells. A number of clinical studies have been preformed using a variety of culture conditions, and several studies are currently in progress that explore the use of ex vivo expanded cells. These studies will be discussed in this review. There are evolving data that suggest that there are real clinical benefits associated with the use of the expanded cells; however, we are still at the early stages of understanding how to optimally culture different cell populations. The next decade should determine what culture conditions and what cell populations are needed for a range of clinical applications.     

Ex vivo amplification of human hematopoietic stem and progenitor cells in an alginate three-dimensional culture system

Introduction: One of the key factors critical for a successful human cord blood transplantation in treating patients with hematopoietic disorders is the number of hematopoietic stem/progenitor cells derived from human cord blood. Here, we report an alginate three‐dimensional (3D) culture system for the expansion of CD34⁺ cells in cord blood mononuclear cells (CBMCs). Methods: Cord blood mononuclear cells were isolated from human cord blood and encapsulated in 3D alginate beads. The cells were grown with different concentrations of cytokines. At day 0, 3, 6, 9, and 12, respectively, the percentage of CD34⁺ cells was quantified by flow cytometry. Colony‐forming cell assay was performed to determine the potential of hematopoietic reconstruction of the amplified cells under the 3D culture system. Results: After culturing for 12 days, the CBMCs encapsulated in the 3D alginate beads were amplified 5.89 ± 0.72 fold, CD34⁺ cells increased from 2.60 ± 0.52% to 13.27 ± 2.65%, and the colony‐forming assay showed that the colony‐forming unit‐granulocyte/granulocyte‐macrophage (CFU‐G/GM) increased from 363.34 ± 34.47/10⁵ cells to 3423.33 ± 645.14/10⁵ cells (P < 0.001). In comparison, the conventional two‐dimensional (2D) culture system showed that the CBMCs, CD34⁺ cells and the CFU‐G/GM were 0.68 ± 0.16 fold, 0.45 ± 0.17%, and 532.92 ± 82.97/10⁵ cells, respectively. Conclusion: This study demonstrates a new and efficient method to amplify the CD34⁺ human cord blood hematopoietic stem/progenitor cells in a 3D alginate culture system ex vivo for extended periods while retaining the hematopoietic reconstruction capacity.     

铁过载对脐带血来源的造血干祖细胞及造血支持细胞的作用观察

目的 探讨铁过载对脐带血(UCB)来源的造血干祖细胞及造血支持细胞,尤其是间充质干细胞(MSCs)的损伤作用.方法 体外培养脐带血单个核细胞(UCB-MNCs)和脐带血间充质干细胞(UCB-MSCs),向培养液中添加200 μmol/L的枸橼酸铁胺(FAC) 24 h建立铁过载模型.分为MNCs-CTL组、MNCs-FAC组、MSCs-CTL组、MSCs-FAC组,每组设3个复孔,实验重复3次.检测细胞内活性氧物质(ROS)水平变化、细胞增殖、分化、凋亡以及造血支持作用.结果 对UCB-MNCs进行铁过载,MNCs-FAC组造血集落形成单位(CFU-E、CFU-GM、BFU-E、CFU-mix)计数显著低于MNCs-CTL组(P＜0.05),MNCs-FAC组造血干细胞(CD+34)、髓系造血细胞(CD+33)、红系造血细胞(GlyA+)比例及计数均显著低于MNCs-CTL组(P均＜0.05);MNCs-FAC组的凋亡率高于MNCs-CTL组(P＜0.05).MSCs-FAC组的群体倍增时间明显长于MSCs-CTL组,且其凋亡率亦高于MSCs-CTL组(P＜0.05).结论 铁过载可抑制造血干祖细胞的增殖、分化,诱导其凋亡,也可抑制MSCs的增殖能力,诱导其凋亡,降低其造血支持能力,且此过程中ROS升高.     

Senescence and functional failure in hematopoietic stem cells

Maintaining normal function of hematopoietic stem cells (HSCs) is critical to blood coagulation, oxygen transportation, and host defense against infection. A potential cause of HSC dysfunction is senescence, in which HSCs and progenitor cells suffer from proliferative arrest. Studies on humans and various animal models have indicated that HSCs can become senescent, showing a significant decline in functional ability with increasing age. There are genetic elements mapped to specific chromosomal sites that regulate HSC senescence. These elements may differ among species, strains, and even individuals. HSC senescence and related pathological effects may not be obvious during normal lifetime under most circumstances since individual primitive HSC clones can function long-term to produce progeny that sustain life-long mature blood cell production. Shortening of telomeres at the chromosomal ends could contribute to HSC senescence, especially when HSCs are stressed under certain pathological conditions. Future studies should define the molecular elements that are important in the regulation of HSC senescence.     

Comparative gene expression in hematopoietic progenitor cells derived from embryonic stem cells

OBJECTIVE: The aim of this study was to characterize at the molecular level the hematopoietic progenitor cells derived from rhesus monkey embryonic stem (ES) cell differentiation. MATERIALS AND METHODS: We purified CD34(+) and CD34(+)CD38(-) cells from rhesus monkey ES cell cultures and examined the expression of a variety of genes associated with hematopoietic development, by semiquantitative polymerase chain reaction analysis. For comparison, we examined cell preparations from fresh or cultured rhesus monkey bone marrow (BM) and from mouse ES cells and BM. RESULTS: We observed a high degree of similarity in the expression patterns of these genes, with only a few exceptions. Most notably, the message of the flt3 gene was undetectable in rhesus monkey ES cell-derived CD34(+) and CD34(+)CD38(-) cells, whereas substantial flt3 expression was observed in the corresponding cells from fresh BM and in CD34(+) cells from cultured BM. The integrin alphaL and interleukin-6 (IL-6) receptor genes also were expressed in CD34(+)CD38(-) cells from BM, but there was little or no expression of these genes in CD34(+)CD38(-) cells derived from ES cells. Parallel analyses, using CD34(+)Lin(-) cells derived from murine ES cell cultures, showed no apparent expression of flt3, integrin alphaL, or IL-6 receptor, whereas corresponding cell preparations isolated from mouse BM expressed high levels of all of these genes. CONCLUSIONS: ES cell-derived hematopoietic progenitors, both from the rhesus monkey and from the mouse, exhibited the same alterations in gene expression compared with BM-derived cells from these animals. These observations could reflect the presence of different subpopulations in the cell fractions that were compared, or they may represent altered biologic properties of ES cell-derived hematopoietic stem cells.