Lymphohematopoietic Progenitors Research Articles

Thymic microenvironment's impact on immunosenescence.

Age-related thymic involution is characterized by the loss of T cell development and the supporting epithelial network, which are replaced by adipose tissue. We previously showed that aging functionally impairs lymphohematopoietic progenitor cells, including thymic early T cell progenitors (ETPs), contributing to thymic involution. Considering that the thymic microenvironment is essential for thymocyte incubation, we aimed to investigate its role in age-related thymic involution and the mechanisms underlying these changes. The challenge in studying these processes led us to transplant T cell-depleted fetal thymus tissue into the kidney capsule of aged mice. This model allowed us to identify the mechanisms driving age-related changes in the thymic microenvironment and to assess whether these changes could be reversed. Flow cytometry was used to detect naïve T cells (CD62L+CD44-), including CD4 CD8 double-negative, double-positive, and single-positive T cells. Real-time PCR was used to detect and quantify signal-joint T cell receptor excision circles. We rearranged δRec-ΨJα in murine peripheral blood leukocytes to evaluate the thymic output of newly developed naïve T cells in the mice and gene expression in the thymus. Age-related thymic involution decreased naïve T cells and increased memory T cells, while fetal thymus transplantation improved thymic output and T cell production and reversed the impairment of thymopoiesis due to thymic involution in aged mice. Furthermore, the expression of key cytokines was restored and ETPs in the aged mice showed normal thymic T cell development. Our study suggests that degenerative changes in the thymic microenvironment are the primary cause of thymic dysfunction, leading to immunosenescence associated with age-related thymic involution.

Lympho-Hematopoietic Microenvironments and Fish Immune System.

Simple SummaryTeleost fish, the most abundant group of vertebrates, represent an excellent tool to establish possible correlations between the histological organization of their lymphoid organs and their immunological capacities. This approach allows us to analyze embryonic and larval lymphopoiesis, the remarkable organization of the teleost thymus, the role of the kidney as a true equivalent of the lympho-hematopoietic bone marrow of higher vertebrates, the mechanisms of antigen trapping in both ellipsoids and the so-called melano-macrophage centers (MMCs) and their relation with the generation of memory and the lack of germinal centers, and the extended development of the lymphoid tissue associated to mucosae.In the last 50 years information on the fish immune system has increased importantly, particularly that on species of marked commercial interest (i.e., salmonids, cods, catfish, sea breams), that occupy a key position in the vertebrate phylogenetical tree (i.e., Agnatha, Chondrichtyes, lungfish) or represent consolidated experimental models, such as zebrafish or medaka. However, most obtained information was based on genetic sequence analysis with little or no information on the cellular basis of the immune responses. Although jawed fish contain a thymus and lympho-hematopoietic organs equivalents to mammalian bone marrow, few studies have accounted for the presumptive relationships between the organization of these cell microenvironments and the known immune capabilities of the fish immune system. In the current review, we analyze this topic providing information on: (1) The origins of T and B lymphopoiesis in Agnatha and jawed fish; (2) the remarkable organization of the thymus of teleost fish; (3) the occurrence of numerous, apparently unrelated organs housing lympho-hematopoietic progenitors and, presumably, B lymphopoiesis; (4) the existence of fish immunological memory in the absence of germinal centers.

EXTL3 mutations cause skeletal dysplasia, immune deficiency, and developmental delay.

We studied three patients with severe skeletal dysplasia, T cell immunodeficiency, and developmental delay. Whole-exome sequencing revealed homozygous missense mutations affecting exostosin-like 3 (EXTL3), a glycosyltransferase involved in heparan sulfate (HS) biosynthesis. Patient-derived fibroblasts showed abnormal HS composition and altered fibroblast growth factor 2 signaling, which was rescued by overexpression of wild-type EXTL3 cDNA. Interleukin-2-mediated STAT5 phosphorylation in patients' lymphocytes was markedly reduced. Interbreeding of the extl3-mutant zebrafish (box) with Tg(rag2:green fluorescent protein) transgenic zebrafish revealed defective thymopoiesis, which was rescued by injection of wild-type human EXTL3 RNA. Targeted differentiation of patient-derived induced pluripotent stem cells showed a reduced expansion of lymphohematopoietic progenitor cells and defects of thymic epithelial progenitor cell differentiation. These data identify EXTL3 mutations as a novel cause of severe immune deficiency with skeletal dysplasia and developmental delay and underline a crucial role of HS in thymopoiesis and skeletal and brain development.

A functional role for the histone demethylase UTX in normal and malignant hematopoietic cells

Ubiquitously transcribed tetratricopeptide repeat, X chromosome (UTX), an H3K27Me2/3 demethylase, has been implicated in development, self-renewal, and differentiation of various organs and embryonic stem cells through chromatin modifications and transcriptional regulation of important developmentally related genes, such as Hox genes. However, the function of UTX in hematopoiesis is not well understood. To study the role of UTX in the mammalian hematopoietic system, we used lentiviral short hairpin RNA constructs to knockdown UTX in the murine hematopoietic progenitor cell line EML, in primary murine bone marrow cells and in leukemic cell lines. We report that Utx is highly expressed in the hematopoietic compartment and that it plays an important role in cell proliferation and homeostasis of hematopoietic cells in vitro. Knockdown of UTX in EML and primary murine bone marrow cells impairs their colony-forming ability. Moreover, knockdown of UTX affects expression of key genes that regulate hematopoietic differentiation such as Mll1, Runx1, and Scl in primary murine bone marrow cells. And we further demonstrate that UTX directly associates with the promoters of the Mll1, Runx1, and Scl genes and modulate their transcription by controlling H3K27me3 marks on respective promoter regions. In addition, UTX depletion severely impaired proliferation of several human leukemia cell lines. Together, these data demonstrate a functional role for UTX in normal and malignant hematopoiesis.

Threshold levels of Flt3‐ligand are required for the generation and survival of lymphoid progenitors and B‐cell precursors

The generation of B-cell precursors (BCP) from lymphohematopoietic progenitors (LHP) in bone marrow is dependent on signals provided by the receptor tyrosine kinase Flt3 and its ligand, Flt3-ligand (FL). Mice deficient in FL exhibit striking reductions in LHP and BCP. Currently, the mechanism by which Flt3 regulates lymphoid lineage/B-cell development is unknown. Here, we show that haploinsufficiency of FL (FL(+/) (-) ) reduced the numbers of LHP, common lymphoid progenitors, and pro-B cells, suggesting that FL levels set a threshold for B lymphopoiesis. Limiting dilution analysis confirmed reduced BCP frequency in FL(+/) (-) mice. Real-time PCR of LHP from FL(+/) (-) animals showed increased transcripts of the B lineage inhibitor id1. However, targeted deletion of id1 did not restore the lymphoid/B lineage deficiencies in FL(-/-) mice, supporting Id1-independent mechanisms. BrdU incorporation studies established that FL is not essential for the proliferation of Flt3(+) multipotential progenitors. Analysis of FL(-/-) progenitors expressing low levels of Flt3 revealed decreased levels of the pro-survival factor Mcl1. Consequently, the Flt3(+) LHP progeny of Flt3(low) LSK(+) cells exhibited increased Annexin V staining. Together, these data suggest that Flt3 signaling initiates a cascade of events in Flt3(low) precursors that promote the survival of LHP from which BCP are derived.

Development of natural killer cells from lymphohematopoietic progenitors of murine fetal liver.

We established a clonal culture system which supports the growth of murine immature natural killer (NK) cells. When we plated day 14 fetal thymocytes in methylcellulose media containing interleukin 2 (IL-2), IL-7 and steel factor (SF), we observed diffuse colonies which could not be classified into known colony types. Cells in the colonies were blast-like and expressed Thy-1 and CD25 but not lineage-specific markers. Cells in the colonies developed into NK1.1+ cells in fetal thymus organ culture indicating that the colonies consist of immature NK cells. We then examined the colony-forming ability of fetal liver cells. The combination of IL-2, IL-7 and SF with or without IL-11 supported formation of few immature NK cell colonies from purified progenitors. Interestingly, addition of IL-11 to the culture stimulated formation of mixed colonies consisting of immature NK cells, B cells, macrophages and/or mast cells. The clonal origin of the mixed NK cell colonies was confirmed by micromanipulation of the colony-forming cells. This culture assay should facilitate the analysis of the pathway and cytokine regulation of NK cell development.

Expression of AA4.1 marks lymphohematopoietic progenitors in early mouse development

The hematopoietic system of mice is established during the early to midgestational stage of development. However, the earliest lymphohematopoietic progenitors that appear during mouse development have been less well characterized compared with the hematopoietic stem cell compartment of fetal liver and bone marrow. We isolated the earliest lymphohematopoietic progenitors by using embryonic stem (ES) cell culture in vitro. Cells with the c-Kit(+)Lin(-) cell surface phenotype were present abundantly in ES cells cocultured with stromal cell lines. We further separated the cells into two distinct cell subsets based on AA4.1 expression. Although AA4.1(+) and AA4.1(-) cells had equivalent potency to generate myeloid cell lineages, the lymphoid potential in ES-cell-derived cells was largely restricted to the cells expressing AA4.1. The same cell type was present abundantly in the early yolk sac and in fewer numbers (approximately 5% of that in the yolk sac) in the caudal half of the developing embryos. These data suggest that AA4.1 is a cell surface marker that can identify the earliest lymphohematopoietic progenitors in mouse development.

Age-Related Perturbation of Thymic Epithelial Cells (TEC) Homeostasis Is Caused by Increased TNFR/Fas-Mediated Apoptosis Coupled with Decreased Proliferation

Age-related thymic involution contributes to severe and clinically significant immune deficiency in the elderly. Proposed mechanisms of age-related thymic involution have focused on either intrinsic defects of lymphohematopoietic progenitors or primary defects of the thymic microenvironment with aging. Previously, we have demonstrated that keratinocyte growth factor (KGF) transiently reverses murine age-related thymic involution by regenerating the TEC compartment. We sought to understand the mechanisms of age-related loss of TECs and to evaluate a potential therapeutic strategy to durably regenerate the thymus. We found a significant increase in the frequency of apoptotic TECs (CD45- MHCII+) coupled with a decline in the frequency of S-phase TECs in young versus aged mice (11 vs 19% and 35% vs 17%, respectively). Telomere shortening was observed in both aged thymocytes and thymic stromal cells, indicating increased replicative senescence in both compartments. Previous studies have demonstrated lack of age-related thymic involution in Fas−/− mice. We found that the frequency of Fas+ TECs was 3-fold higher in aged mice compared to young mice. A distinct TEC subpopulation expressed Fas, but there was no increase in Fas+ CD45+ lymphoid cells. Although FasL was not expressed by thymocytes, expression of FasL by intrathymic mature T cells (ITMTs, CD4+CD8−CD44high or CD4−CD8+CD44high) increased 2-fold in aged mice. In contrast, there was no change in the frequency of FasL+ splenic memory T cells. To understand how Fas expression is induced in aged TEC, we examined the effects of two pro-inflammatory cytokines, TNF-α and IL-1β, which are known to induce Fas expression. Both TNF-α and IL-1β up-regulated Fas expression on K5+K8+ (possible precursor), K5−K8+ (cortical), or K5+K8− (medullary) TEC clones derived from young mice. RT-PCR and FACS analyses showed that the aged thymus had 4–5-fold higher levels of TNF-α, and TNF-α expression was increased in aged CD4+ and CD8+ ITMT, as well as all subsets of thymocytes. To elucidate the molecular mechanisms of apoptosis in the aged TEC, we analyzed levels of activated caspase-8, a key mediator of TNFR/Fas-induced apoptosis. Almost 50% of TECs in aged mice but only 25% in young mice had activated caspase-8. Furthermore, in vivo treatment (0.4mg/mouse, every 3rd day for 1 month) of a caspase-8-specific inhibitor (z-IETD-fmk) increased the number of TECs, decreased TEC apoptosis, and enhanced thymopoiesis in aged mice. The data suggest that thymic aging is mediated by decreased regenerative potential of TEC coupled with increased expression of inflammatory cytokines by both ITMT and thymocytes that make TECs more susceptible to apoptosis by either inducing Fas expression on TECs or directly triggering TNFR-mediated apoptosis. The inflammatory effects of ITMT on TEC death provides a feed-forward mechanism by which peripheral memory T-cell generation and immune senescence is linked to thymic involution.

The Renin-Angiotensin Axis Regulates the Development of a Yolk Sac-Like Hemangioblastic Progenitor of Primitive and Definitive Hematopoiesis from Human Pluripotent Stem Cells.

We have defined serum-free (SF) culture conditions for efficiently generating clonogenic hemangioblasts from pluripotent human embryonic stem cells (hESC). We also report that angiotensin-converting enzyme (ACE), a critical physiologic regulator of blood pressure, angiogenesis, and inflammation, is a novel marker for identifying and purifying hemangioblastic stem-progenitors from differentiating hESC. Using in vitro clonal assays of primitive and definitive hematopoietic potential, we demonstrated that ACE+ human embryoid body (hEB) cell populations contain a common yolk sac (YS)-like progenitor for not only endothelium, but also both primitive and definitive hematopoieses. Thrombopoietin (TPO) and basic fibroblast growth factor (FGF2) were identified as critical growth factors for expanding ACE+ hemangioblasts. ACE+CD34-CD45- human embryoid body (hEB) cells contained the highest number of these progenitors. Further maturation of ACE+ hemangioblasts in a stromal environment produced definitive-type lympho-hematopoietic progenitors, albeit with a late YS, and not AGM phenotype. The developmental kinetics of YS-like hematopoiesis generated from hEB cells was remarkably in parallel to the actual timeline of human YS development, which occurs during weeks 2–6 of human gestation. We also showed that ACE and the renin-angiotensin system (RAS) axis plays a direct role in hESC-derived hemangioblastic differentiation, and is directly regulated via signaling through the two major angiotensin peptide receptors (AGTR1 and AGTR2). Moreover, directed differentiation of hemangioblasts toward either endothelium, or multipotent primitive hematopoietic progenitors was efficiently enhanced via modulation of either AGTR1 or AGTR2 signaling. RAS axis manipulation may therefore be exploited for directing the differentiation of transplantable hESC-derived hemangioblastic stem-progenitors, thus providing novel opportunities for human tissue engineering. The key molecular and cellular events of human hemato-endothelial genesis can now be delineated in a manner that was previously impossible due to inaccessibility of human embryonic tissue. [Display omitted]

The aged thymus shows normal recruitment of lymphohematopoietic progenitors but has defects in thymic epithelial cells

Aging is associated with reduced numbers of all thymocyte sub-populations, including early T-cell progenitors. However, it is unclear if this is due to inadequate recruitment of lymphohematopoietic progenitor cells (LPCs) to the aged thymus or to abnormal development of T cells within the thymus. We found that LPCs from young mice were recruited equally well to the thymi of young or aged mice and that thymic stromal cells (TSCs) from young and old mice expressed similar levels of P-selectin and CCL25, which are believed to mediate recruitment of LPCs to the adult thymus. However, the number of recruited thymocytes in old thymus was markedly reduced after two weeks, indicating that T-cell development or proliferation is defective in the aged thymus. We also found that LPCs from aged and young mice have similar capacities to seed a fetal thymus that was transplanted under the kidney capsule. Thymic epithelial cells (TECs) in aged mice had lower proliferative capacity and higher rate of apoptosis, compared with findings in young animals. In addition, immunofluorescence staining with antibodies to cortical and medullary TECs revealed that aged thymi had a disorganized thymic stromal architecture, combined with reduced cellularity of the medulla, and apoptosis of thymocyte sub-populations in the medullary microenvironment was increased, compared with that in young mice. We conclude that aging does not impair recruitment of LPCs to the thymus, but is characterized by abnormalities in thymic epithelial architecture, especially medullary TEC function that may provide sub-optimal support for thymic development of LPCs.

Lymphohematopoietic progenitors do not have a synchronized defect with age‐related thymic involution

It has been speculated that aging lymphohematopoietic progenitor cells (LPC) including hematopoietic stem cells (HSC) and early T-cell progenitors (ETP) have intrinsic defects that trigger age-related thymic involution. However, using a different approach, we suggest that that is not the case. We provided a young thymic microenvironment to aged mice by transplanting a fetal thymus into the kidney capsule of aged animals, and demonstrated that old mouse-derived LPCs could re-establish normal thymic lymphopoiesis and all thymocyte subpopulations, including ETPs, double negative subsets, double positive, and CD4(+) and CD8(+) single positive T cells. LPCs derived from aged mice could turn over young RAG(-/-) thymic architecture by interactions, as well as elevate percentage of peripheral CD4(+)IL-2(+) T cells in response to costimulator in aged mice. Conversely, intrathymic injection of ETPs sorted from young animals into old mice did not restore normal thymic lymphopoiesis, implying that a shortage and/or defect of ETPs in aged thymus do not account for age-related thymic involution. Together, our findings suggest that the underlying cause of age-related thymic involution results primarily from changes in the thymic microenvironment, causing extrinsic, rather than intrinsic, defects in T-lymphocyte progenitors.

Sustained thymopoiesis and improvement in functional immunity induced by exogenous KGF administration in murine models of aging

AbstractAge-related thymopoietic insufficiency has been proposed to be related to either defects in lymphohematopoietic progenitors or the thymic microenvironment. In this study, we examined whether keratinocyte growth factor (KGF), an epithelial cell–specific growth factor, could increase thymopoietic capacity in aged mice by restoration of the function of thymic epithelial cells (TECs). The thymic cellularity in KGF-treated aged mice increased about 4-fold compared to placebo-treated mice, resulting in an equivalent thymic cellularity to young mice. Enhanced thymopoiesis was maintained for about 2 months after a single course of KGF, and sustained improvement was achieved by administration of monthly courses of KGF. With the enhanced thymopoiesis after KGF treatment, the number of naive CD4 T cells in the periphery and T-cell–dependent antibody production improved in aged mice. KGF induced increased numbers of TECs and intrathymic interleukin-7 (IL-7) production and reorganization of cortical and medullary architecture. Furthermore, KGF enhanced thymopoiesis and normalized TEC organization in klotho (kl/kl) mice, a model of premature degeneration and aging, which displays thymopoietic defects. The result suggests that TEC damage is pathophysiologically important in thymic aging, and KGF therapy may be clinically useful in improving thymopoiesis and immune function in the elderly.

Angiotensin-Converting Enzyme (ACE) Expression Defines the Earliest Primitive and Definitive Lympho-Hematopoietic Progenitors Derived from Human Embryonic Stem Cells.

Pluripotent human embryonic stem cells (hESC) provide unprecedented opportunities for studying obscure human developmental events, such as those required for the genesis of hematopoietic stem cells (HSC).A poorly characterized aspect of embryonic and adult HSC development is the role of the renin-angiotensin system (RAS), which is implicated in regulating HSC proliferation at yolk sac (YS), fetal, and adult stages. We have recently described somatic ACE surface expression not only in adult HSC, but also at the earliest stages of emergent hemato-endotheliogenesis using a novel monoclonal antibody (BB9). ACE expression identifies primitive subsets of adult CD34+ bone marrow HSC, but more intriguingly, marks emergent hematopoietic cells from both CD34− and CD34+ areas of human YS, intraembryonic subaortic patches, and hemogenic endothelial layers of the aorta-gonad-mesonephros (AGM) region. The pattern of human embryonic ACE expression is consistent with the hypothesis of ACE+CD34− hemangioblasts emigrating dorsally from the para-aortic splanchnopleura, and subsequently colonizing the ventral aorta to give rise to CD34+ hemogenic endothelium.We tested the hypothesis that ACE expression would similarly identify emerging hemato-endothelial progenitors derived from our recently-described hESC-based hematopoietic differentiation system. This embryoid body (hEB)-based system recapitulates hemato-endothelial, primitive, and definitive stages of human embryonic blood development. ACE expression kinetics during hEB differentiation correlated well with the onset of hemato-endothelial differentiation and gene expression (e.g., SCL/tal1, CDX4, CD31, and CD34). Furthermore, using improved, novel methods of hEB hematopoietic differentiation that dramatically augment multilineage progenitors for both primitive and definitive hematopoiesis, we observed that levels of hEB ACE expression were directly correlated to increased hematopoietic potency. Subsequent FACS purification of these hEB cells demonstrated that the earliest detectable multilineage lympho-hematopoietic competency was contained entirely within both ACE+CD34−CD45− hEB and ACE+CD34+CD45− populations. These early ACE+CD45− hEB populations were heterogenous, and co-expressed abundant levels of known hemato-endothelial markers such as CD31, KDR, CD164, CD43, and CD71.Using novel in vitro assays of primitive and definitive hematopoietic potential, we demonstrated that ACE+ hEB contained common progenitors for both primitive and definitive hematopoiesis, with ACE+CD34−CD45− hEB being enriched for the highest number of progenitors. We were also able to demonstrate that further maturation of these ACE+ hEB cells in an in vitro AGM-like stromal environment produced definitive hematopoietic progenitors that resembled those obtained from cord blood CD34+ cells. The regulatory effects of angiotensin agonist/antagonist peptides on hEB-derived hematopoietic ACE+ progenitors, and their in vivo correlation to ACE+ cells obtained from early human YS and AGM tissue is currently in progress. Furthermore, single-cell analysis is underway to delineate an ACE+ hEB-derived hemangioblastic precursor of not only endothelium, but also primitive and definitive lympho-hematopoiesis.

Leukosialin (CD43) defines hematopoietic progenitors in human embryonic stem cell differentiation cultures

AbstractDuring hematopoietic differentiation of human embryonic stem cells (hESCs), early hematopoietic progenitors arise along with endothelial cells within the CD34+ population. Although hESC-derived hematopoietic progenitors have been previously identified by functional assays, their phenotype has not been defined. Here, using hESC differentiation in coculture with OP9 stromal cells, we demonstrate that early progenitors committed to hematopoietic development could be identified by surface expression of leukosialin (CD43). CD43 was detected on all types of emerging clonogenic progenitors before expression of CD45, persisted on differentiating hematopoietic cells, and reliably separated the hematopoietic CD34+ population from CD34+CD43–CD31+KDR+ endothelial and CD34+CD43–CD31–KDR– mesenchymal cells. Furthermore, we demonstrated that the first-appearing CD34+CD43+CD235a+CD41a+/–CD45– cells represent precommitted erythro-megakaryocytic progenitors. Multipotent lymphohematopoietic progenitors were generated later as CD34+CD43+CD41a–CD235a–CD45– cells. These cells were negative for lineage-specific markers (Lin–), expressed KDR, VE-cadherin, and CD105 endothelial proteins, and expressed GATA-2, GATA-3, RUNX1, C-MYB transcription factors that typify initial stages of definitive hematopoiesis originating from endothelial-like precursors. Acquisition of CD45 expression by CD34+CD43+CD45–Lin– cells was associated with progressive myeloid commitment and a decrease of B-lymphoid potential. CD34+CD43+CD45+Lin– cells were largely devoid of VE-cadherin and KDR expression and had a distinct FLT3highGATA3lowRUNX1lowPU1highMPOhighIL7RAhigh gene expression profile.

Hematopoietic Stem Cell Antigen CD34: Role in Adhesion or Homing

CD34 is highly glycosylated surface antigen of enormous clinical utility in the identification, enumeration, and purification of engraftable lymphohematopoietic progenitors for transplantation. However, recently its importance in the specific marking of most immature hematopoietic stem/progenitor cells have been questioned by addressing long-term reconstitution capability of CD34(-) hematopoietic cellular fractions. These controversies have stimulated a demand for elucidation of the structure, function, and molecular interactions of CD34 to define exactly its biological significance in clinical regimens. There is accumulating data showing the participation of CD34 in adhesion or perhaps homing of lymphohematopoietic progenitors. On the other hand, CD34 has been demonstrated to down-regulate cytokine-induced differentiation and proliferation of CD34(+) cells. Studies in CD34 knockout mice revealed normal hematopoiesis but a profound delay in hematopoietic reconstitution after sublethal irradiation of the mice. In short, CD34 expression is likely to represent a specific state of hematopoietic development that may have altered adhering properties with expanding and differentiating capabilities in both in vitro and in vivo conditions. This article focuses on the adhesive properties of CD34 and its potential role in homing, which are likely to mimic lymphocyte homing to the inflammatory sites.

Development of Lymphohematopoietic Progenitors during Human Embryonic Stem (hES) Cell Differentiation on OP9 Stromal Cells.

hES cells provide an unique opportunity to study the earliest stages of hematopoietic commitment which are not easily accessible in the human embryo. To model early hematopoietic development, we cultured H1 and H9 hES cell lines on OP9 stromal cells without the addition of cytokines. On day 2 of co-culture, hES cells up-regulated brachyury expression and began to form mesodermal-like colonies. A transient population of blast colony-forming cells (CFCs) with the potential to differentiate into blood and endothelial cells was detected on days 3–6 of co-culture. CD34+ cells first appeared on day 3–4 of co-culture, which was coincident with induction of the transcription factors GATA-1, GATA-2 and SCL. CD43+ and CD41a+ cells along with CFCs emerged 2 days later within CD34+ population; 3–4 days before the appearance of CD45+ cells. We were able to obtain up to 20% of CD34+ cells from hES/OP9 co-culture and isolate up to 107 CD34+ cells with more than 95% purity from a similar number of initially plated hES cells after 8–9 days of culture. The hES cell-derived CD34+ cells were highly enriched in CFCs, displayed CD90+CD117+CD164+CD38- phenotype of primitive hematopoietic progenitors, and contained ALDHhigh cells as well cells with verapamil-sensitive ability to efflux rhodamine 123. Isolated CD34+ cells differentiated into lymphoid (NK cells) as well as myeloid (neutrophils and macrophages) lineages when cultured on MS-5 stromal cells in the presence of SCF, Flt3-L, IL7 and IL3. These data indicate that hES cell/OP9 co-culture reproduces the major events that are observed during embryonal hematopoietic development, including the formation of lympho-myeloid progenitors. We employed OP9 system for identification of the phenotype of early hematopoietic progenitors in humans and to directly differentiate hES cells into different blood lineages.

Combination of 3′ and 5′ IgH regulatory elements mimics the B-specific endogenous expression pattern of IgH genes from pro-B cells to mature B cells in a transgenic mouse model

To ensure the B cell differentiation stage specificity of the intronic Eμ element and of the locus control region (LCR) that lies downstream of the IgH chain locus, we generated transgenic mice harboring a V H promoter-GFP reporter gene linked to the 3′LCR region and the Eμ element. By flow cytometry, GFP + lymphocytes were observed amongst pro-B cells (B220 +CD43 +CD117 +) and at all stages of differentiation up to mature B cells (B220 +IgM +IgD +). Expression was strictly confined to cells committed to the B lymphocyte lineage as judged by the lack of GFP +Thy1,2 + cells (T lymphocytes) and GFP +B220 −CD117 +CD43 + cells (uncommitted lymphohematopoietic progenitors). Therefore, the Eμ-GFP-3′LCR transgene is not expressed by hematopoietic stem cells, begins its expression in pro-B cells and is specifically active at all stages of B cell maturation. The combination of 3′ and 5′ IgH regulatory elements thus appears as a potentially useful cassette in transgenes that require a stringent and early B lineage-specific expression.

Long-lived polyclonal B-cell lines derived from midgestation mouse embryo lymphohematopoietic progenitors reconstitute adult immunodeficient mice.

Lymphohematopoietic progenitors derived from midgestation mouse embryos were established in long-term cultures with stromal cell monolayers and interleukin 7 (IL-7), giving rise to B-lineage cell lines. The initial emergence and in vitro establishment of these early embryo cell lines were highly sensitive to IL-7-mediated signals, in comparison to cell lines similarly obtained using precursors from late fetal liver (> 13 days postcoitum) and adult bone marrow. The early embryo-derived progenitors spontaneously differentiated in vitro to CD19(+)IgM(+) immature B cells in the presence of optimal concentrations of IL-7, in contrast to those progenitors obtained from late gestation and adult mice, whose differentiation only occurred in the absence of IL-7. The newly in vitro-generated B cells of the early embryo cell lines repopulated adult immunodeficient severe combined immunodeficient mice on their adoptive transfer in vivo and generated specific humoral immune responses after immunization.

Vascular endothelial growth factor synergistically enhances bone morphogenetic protein-4-dependent lymphohematopoietic cell generation from embryonic stem cells in vitro

AbstractThe totipotent mouse embryonic stem (ES) cell is known to differentiate into cells expressing the β-globin gene when stimulated with bone morphogenetic protein (BMP)-4. Here, we demonstrate that BMP-4 is essential for generating both erythro-myeloid colony-forming cells (CFCs) and lymphoid (B and NK) progenitor cells from ES cells and that vascular endothelial growth factor (VEGF) synergizes with BMP-4. The CD45+ myelomonocytic progenitors and Ter119+ erythroid cells began to be detected with 0.5 ng/mL BMP-4, and their levels plateaued at approximately 2 ng/mL. VEGF alone weakly elevated the CD34+ cell population though no lymphohematopoietic progenitors were induced. However, when combined with BMP-4, 2 to 20 ng/mL VEGF synergistically augmented the BMP-4-dependent generation of erythro-myeloid CFCs and lymphoid progenitors from ES cells, which were enriched in CD34+ CD31lo and CD34+CD45− cell populations, respectively, in a dose-dependent manner. Furthermore, during the 7 days of in vitro differentiation, BMP-4 was required within the first 4 days, whereas VEGF was functional after the action of BMP-4 (in the last 3 days). Thus, VEGF is a synergistic enhancer for the BMP-4-dependent differentiation processes, and it seems to be achieved by the ordered action of the 2 factors.

Vascular endothelial growth factor synergistically enhances bone morphogenetic protein-4-dependent lymphohematopoietic cell generation from embryonic stem cells in vitro

The totipotent mouse embryonic stem (ES) cell is known to differentiate into cells expressing the β-globin gene when stimulated with bone morphogenetic protein (BMP)-4. Here, we demonstrate that BMP-4 is essential for generating both erythro-myeloid colony-forming cells (CFCs) and lymphoid (B and NK) progenitor cells from ES cells and that vascular endothelial growth factor (VEGF) synergizes with BMP-4. The CD45+ myelomonocytic progenitors and Ter119+ erythroid cells began to be detected with 0.5 ng/mL BMP-4, and their levels plateaued at approximately 2 ng/mL. VEGF alone weakly elevated the CD34+ cell population though no lymphohematopoietic progenitors were induced. However, when combined with BMP-4, 2 to 20 ng/mL VEGF synergistically augmented the BMP-4-dependent generation of erythro-myeloid CFCs and lymphoid progenitors from ES cells, which were enriched in CD34+ CD31lo and CD34+CD45− cell populations, respectively, in a dose-dependent manner. Furthermore, during the 7 days of in vitro differentiation, BMP-4 was required within the first 4 days, whereas VEGF was functional after the action of BMP-4 (in the last 3 days). Thus, VEGF is a synergistic enhancer for the BMP-4-dependent differentiation processes, and it seems to be achieved by the ordered action of the 2 factors.