In vitro and in vivo evidence for the long-term multilineage (myeloid, B, NK, and T) reconstitution capacity of ex vivo expanded human CD34(+) cord blood cells

The aim of the present report is to describe clinically relevant culture conditions that support the expansion of primitive hematopoietic progenitors/stem cells, with maintenance of their hematopoietic potential as assessed by in vitro assays and the NOD-SCID in vivo repopulating capacity.CD34(+) cord blood (CB) cells were cultured in serum-free medium containing stem cell factor, Flt3 ligand, megakaryocyte growth and development factor, and granulocyte colony-stimulating factor. After 14 days, the primitive functions of expanded and nonexpanded cells were determined in vitro using clonogenic cell (colony-forming cells, long-term culture initiating cell [LTC-IC], and extended [E]-LTC-IC) and lymphopoiesis assays (NK, B, and T) and in vivo by evaluating long-term engraftment of the bone marrow of NOD-SCID mice. The proliferative potential of these cells also was assessed by determining their telomere length and telomerase activity.Levels of expansion were up to 1,613-fold for total cells, 278-fold for colony-forming unit granulocyte-macrophage, 47-fold for LTC-IC, and 21-fold for E-LTC-IC. Lymphoid B-, NK, and T-progenitors could be detected. When the expanded populations were transplanted into NOD-SCID mice, they were able to generate myeloid progenitors and lymphoid cells for 5 months. These primitive progenitors engrafted the NOD-SCID bone marrow, which contained LTC-IC at the same frequency as that of control transplanted mice, with conservation of their clonogenic capacity. Moreover, human CD34(+)CDl9(-) cells sorted from the engrafted marrow were able to generate CD19(+) B-cells, CD56(+)CD3(-) NK cells, and CD4(+)CD8(+)alphabetaTCR(+) T-cells in specific cultures. Our expansion protocol also maintained the telomere length in CD34(+) cells, due to an 8.8-fold increase in telomerase activity over 2 weeks of culture.These experiments provide strong evidence that expanded CD34(+) CB cells retain their ability to support long-term hematopoiesis, as shown by their engraftment in the NOD-SCID model, and to undergo multilineage differentiation along all myeloid and the B-, NK, and T-lymphoid pathways. The expansion protocol described here appears to maintain the hematopoietic potential of CD34(+) CB cells, which suggests its relevance for clinical applications.     

Lymphoid progenitors in normal mouse lymph nodes develop into NK cells and T cells in vitro and in vivo

We have identified a population of normal mouse LN cells, termed LN lymphoid progenitor (LNLP), resembling common lymphoid progenitor (CLP) in the bone marrow. LNLPs lack lineage markers and express CD127, low levels of CD117 (c-Kit), and Sca-1, but lack fms-related tyrosine kinase 3. They efficiently differentiate in vitro into natural killer (NK) cells and T cells, but not mature B cells. LNLPs injected into nonirradiated lymphopenic mice that have no LN develop into mostly splenic T cells with low numbers of NK cells and B cells. When injected into irradiated mice, they generate NK cells and T cells, but not B cells, in the LN. By contrast, bone marrow CLPs develop into mostly B cells with very small numbers of T and NK cells in recipients' spleen and LN. LNLPs have NK and T-cell potentials, but little B-cell potential, and they can develop into NK cells within the LN of normal mice, but their contribution to the T-cell lineage is unknown.     

Distinct roles of IL-7 and stem cell factor in the OP9-DL1 T-cell differentiation culture system

OBJECTIVE: The OP9-DL1 culture system is an in vitro model for T-cell development in which activation of the Notch pathway by Delta-like 1 promotes differentiation of mature T cells from progenitors. The roles of specific cytokines in this culture system have not been well defined, and controversy regarding the role of IL-7 has recently emerged. We examined the roles played by IL-7, Flt3 ligand, and stem cell factor (SCF) in differentiation of adult bone marrow cells in the OP9-DL1 culture system. METHODS: Hematopoietic progenitor cells isolated from mouse bone marrow were cultured with OP9 or OP9-DL1 stromal cells and evaluated for T and B lymphocyte differentiation using immunofluorescent staining. RESULTS: IL-7 provided both survival/proliferation and differentiation signals in a dose-dependent manner. T-cell development from the CD4/CD8 double-negative (DN) stage to the CD4/CD8 double-positive (DP) stage required IL-7 provided by the stromal cells, while differentiation from the DP to the CD8 single-positive (SP) stage required addition of exogenous IL-7. SCF favored the proliferation of DN lymphoid progenitors and inhibited differentiation to the DP stage in a dose-dependent manner. Conversely, blocking the function of SCF expressed endogenously by OP9-DL1 cells inhibited proliferation of lymphoid progenitors and accelerated T-lineage differentiation. Flt3 ligand promoted proliferation without affecting differentiation. CONCLUSION: These results validate the OP9-DL1 model for the analysis of T-cell development from bone marrow-derived progenitor cells, and demonstrate specific roles of SCF, IL-7, and Flt3L in promoting efficient T-lineage differentiation.     

Isolation and characterization of plasmacytoid dendritic cells from Flt3 ligand and granulocyte-macrophage colony-stimulating factor-treated mice.

Interferon alpha/beta plays an important role in the first-line defense against viral infections and can modulate cytokine responses by T-helper cells. Type 1 interferons (IFNs) are clinically important in infectious diseases and in the treatment of leukemia and lymphomas. Many different cell types have the capacity to produce IFN-alpha after encounter with virus and bacteria. The major, natural type 1 IFN-producing cell in humans was recently described as the plasmacytoid T cell, or pDC2, and it can differentiate into dendritic cells (DCs) on culture. This study describes the murine natural IFN-alpha-producing cell, or pDC2, that shares morphologic features with its human counterpart but has some distinct phenotypical characteristics. Murine plasmacytoid DCs can be differentially isolated based on their expression of CD11c, B220 (CD45R), and Thy1.2 (CD90). They lack expression of myeloid (eg, CD11b) antigens and CD8 alpha, a marker used to isolate lymphoid DCs. Like human pDC2, murine plasmacytoid DCs exhibit their maximal type 1 IFN-producing capacity at a precursor stage; pDCs isolated from bone marrow responded to viral stimulation with higher IFN-alpha production than cells of the same phenotype isolated from spleen. Mobilization of mice with Flt3 ligand (Flt3L) or Flt3L and granulocyte-macrophage colony-stimulating factor, hematopoietic factors that specifically enhance DC growth, resulted in strikingly increased numbers of pDC in bone marrow and spleen. The isolation of this novel murine DC subset may serve as a useful tool in the study of viral immunobiology and for the design of treatments for murine malignancies.     

Presence of primitive lymphoid progenitors with NK or B potential in ex vivo expanded bone marrow cell cultures

OBJECTIVE: In previous work, we showed that CD34+ bone marrow cells can be successfully expanded along the myeloid pathway in stroma- and serum-free conditions in the presence of SCF+IL-3+IL-6+Flt3-l+G-CSF+MGDF. Due to the lack of phenotypically detectable lymphoid cells, it was necessary to address the question of the lymphoid potential of the expanded populations under these conditions. MATERIALS AND METHODS: The present report describes a long-term culture system that supports human B- and NK-cell differentiation from the day 14 fraction without further selection of the more primitive cells. In NK proliferation assays, the cells were maintained over stroma cells in the presence of IL-2 for 4-5 weeks. NK initiating cells (NK-IC) were determined by a limiting dilution assay. In B-cell cultures, the expanded cells were maintained over MS5 in the presence of Flt3-l for 4-8 weeks. RESULTS: NK cells rose from 0.2%+/-0.04% at culture initiation to 71%+/-6% at week 5. These cells displayed cytolytic activity. NK-IC evaluation showed a mean 18-fold expansion in the day 14 expanded fraction as compared to the initial day 0 fraction. Similarly, CD19+ cells rose from 0.1% at culture initiation to 30%+/-1% at week 6. Cells produced under these B-LTC conditions were CD34-CD19+CD10+. We also demonstrated that the CD34+/Lin- sorted cells from the day 14 fraction gave rise to NK and B cells. CONCLUSION: This culture system permits the revelation of a population that, although poorly represented in terms of phenotypically detectable cells, nevertheless retains high levels of lymphoid NK and B potential after 14 days expansion. Such data suggest the persistence, or expansion, of lymphoid progenitors and, hence, the multipotentiality of the expanded progenitor/stem cells.     

Molecular cytogenetics of stem cells: target cells of chromosome aberrations as revealed by the application of fluorescence in situ hybridization to fluorescence-activated cell sorting

The lineage involvement in stem cell disorders, such as chronic myeloid leukemia (CML) and myelodysplastic syndrome (MDS), remains unclear. To explore this issue, we used fluorescence in situ hybridization for cells sorted by fluorescence-activated cell sorting (FACS) from 12 patients with chronic-phase CML. Philadelphia chromosome (Ph) was found in pluripotent stem cells (CD34+Thy-1+), B cells (CD34+CD19+), and T/natural killer (NK) progenitor cells (CD34+CD7+) collected by FACS from bone marrow cells. B (CD19+), T (CD3+), and NK (CD3-CD56+) cells showed a marked decrease in Ph+ cells between progenitor cells and mature cells The Ph+ T and NK cells decreased to below background levels. These data suggest that Ph+ lymphocytes either do not differentiate or are eliminated during their maturation process Among 7 MDS patients associated with trisomy 8, sorted lymphocytes from peripheral blood did not have +8. CD34+ subpopulations from bone marrow including B,T/NK progenitors, and pluripotent progenitor cells also did not have +8.Trisomy 8 was identified from the level of multipotent colony-forming units (CD34+CD33+), and the lymphoid lineage was not involved. Thus, MDS with trisomy 8 conceivably arises from nonlymphoid progenitor cells, sparing T, B, or NK cells. Further studies using molecular cytogenetics may clarify the mechanism of leukemia happening at the level of stem cells.     

A murine Mll-AF4 knock-in model results in lymphoid and myeloid deregulation and hematologic malignancy

The 2 most frequent human MLL hematopoietic malignancies involve either AF4 or AF9 as fusion partners; each has distinct biology but the role of the fusion partner is not clear. We produced Mll-AF4 knock-in (KI) mice by homologous recombination in embryonic stem cells and compared them with Mll-AF9 KI mice. Young Mll-AF4 mice had lymphoid and myeloid deregulation manifest by increased lymphoid and myeloid cells in hematopoietic organs. In vitro, bone marrow cells from young mice formed unique mixed pro-B lymphoid (B220(+)CD19(+)CD43(+)sIgM(-), PAX5(+), TdT(+), IgH rearranged)/myeloid (CD11b/Mac1(+), c-fms(+), lysozyme(+)) colonies when grown in IL-7- and Flt3 ligand-containing media. Mixed lymphoid/myeloid hyperplasia and hematologic malignancies (most frequently B-cell lymphomas) developed in Mll-AF4 mice after prolonged latency; long latency to malignancy indicates that Mll-AF4-induced lymphoid/myeloid deregulation alone is insufficient to produce malignancy. In contrast, young Mll-AF9 mice had predominately myeloid deregulation in vivo and in vitro and developed myeloid malignancies. The early onset of distinct mixed lymphoid/myeloid lineage deregulation in Mll-AF4 mice shows evidence for both "instructive" and "noninstructive" roles for AF4 and AF9 as partners in MLL fusion genes. The molecular basis for "instruction" and secondary cooperating mutations can now be studied in our Mll-AF4 model.     

The Hedgehog receptor Patched controls lymphoid lineage commitment

A first step in hematopoiesis is the specification of the lymphoid and myeloid lineages from multipotent progenitor cells in the bone marrow. Using a conditional ablation strategy in adult mice, we show that this differentiation step requires Patched (Ptch), the cell surface-bound receptor for Hedgehog (Hh). In the absence of Ptch, the development of T- and B-lymphoid lineages is blocked at the level of the common lymphoid progenitor in the bone marrow. Consequently, the generation of peripheral T and B cells is abrogated. Cells of the myeloid lineage develop normally in Ptch mutant mice. Finally, adoptive transfer experiments identified the stromal cell compartment as a critical Ptch-dependent inducer of lymphoid versus myeloid lineage commitment. Our data show that Ptch acts as a master switch for proper diversification of hematopoietic stem cells in the adult organism.     

Overexpression of murine TSLP impairs lymphopoiesis and myelopoiesis

The role of thymic stromal cell-derived lymphopoietin (TSLP) in regulating hematopoiesis is poorly characterized, so we investigated its regulatory effects in vivo using TSLP transgenic mice. Overexpression of TSLP disrupted hematopoietic homeostasis by causing imbalances in lymphopoiesis and myelopoiesis. Mice harboring a TSLP transgene had 5- to 700-fold fewer B and T precursors and no detectable pre-B lymphocyte colonyforming activity in the marrow or spleen. Conversely, TSLP transgenic mice possessed 15 to 20 times more splenic myeloid precursors than their littermates, and progenitor activity of the granulocyteerythrocyte-macrophage-megakaryocyte colony-forming units was significantly elevated. The arrest in lymphopoiesis and the expansion of myeloid progenitor cells in TSLP transgenic mice suggest that TSLP has negative and positive regulatory effects on lymphoid and myeloid development, respectively.     

Reconstitution of early lymphoid proliferation and immune function in Jak3-deficient mice by interleukin-3.

Expansion of early lymphoid progenitors requires interleukin-7 (IL-7), which functions through gamma(c)-mediated receptor activation of Jak3. Jak3 deficiency is a cause of severe combined immunodeficiency (SCID) in humans and mice. IL-3 activates many of the same signaling pathways as IL-7, such as Stat5, but achieves this effect through the activation of Jak2 rather than Jak3. We hypothesized that expansion of an IL-7-responsive precursor population through a Jak3-independent pathway using IL-3 may stimulate early lymphoid progenitors and restore lymphopoiesis in Jak3(-/-) mice. Newborn Jak3(-/-) mice that were injected with IL-3 demonstrated thymic enlargement, a 2- to 20-fold increase in thymocyte numbers, and up to a 10-fold expansion in the number of CD4(+), CD8(+), and B220(+)/IgM(+) splenic lymphocytes, consistent with an effect upon an early lymphoid progenitor population. In contrast to control mice, IL-3-treated Jak3(-/-) mice challenged with the allogeneic major histocompatibility complex (MHC) class I-bearing tumor P815 developed a specific CD8-dependent cytotoxic T lymphocyte (CTL) response. IL-3-treated mice also mounted influenza-specific CTL responses and survival was prolonged. The beneficial effects of IL-3 are proposed to be produced by stimulation of a lymphoid precursor population of IL-7Ralpha(+)/IL-3Ralpha(+) cells that we identified in wild-type bone marrow. In vitro, we show that an early IL-7R(+) lymphoid progenitor population expresses IL-3R and proliferates in response to IL-3 and that IL-3 activates Stat5 comparably to IL-7. Clinically, IL-3 may therefore be useful treatment for X-linked and Jak3-deficient SCID patients who lack bone marrow donors.     

Flk2+ myeloid progenitors are the main source of Langerhans cells

Langerhans cells (LCs) are antigen-presenting cells (APCs) residing in the epidermis that play a major role in skin immunity. Our earlier studies showed that when skin is inflamed LCs are replaced by bone marrow-derived progenitor cells, while during steady-state conditions LCs are able to self-renew in the skin. Identification of the LC progenitors in bone marrow would represent a critical step toward identifying the factors that regulate LC generation as well as their trafficking to the skin. To determine LC lineage origin, we reconstituted lethally irradiated CD45.2 mice with rigorously purified lymphoid and myeloid progenitors from CD45.1 congenic mice. Twenty-four hours later, we exposed the mice to UV light to deplete resident LCs and induce their replacement by progenitors. Reconstitution with common myeloid progenitors (CMPs), common lymphoid progenitors (CLPs), granulocyte-macrophage progenitors (GMPs), or early thymic progenitors led to LC generation within 2 to 3 weeks. CMPs were at least 20 times more efficient at generating LCs than CLPs. LCs from both lineages were derived almost entirely from fetal liver kinase-2+ (Flk-2+) progenitors, displayed typical dendritic-cell (DC) morphology, and showed long-term persistence in the skin. These results indicate that LCs are derived mainly from myeloid progenitors and are dependent on Flt3-ligand for their development.     

The heparin binding PECAM-1 adhesion molecule is expressed by CD34+ hematopoietic precursor cells with early myeloid and B-lymphoid cell phenotypes

The platelet-endothelial cell adhesion molecule-1 (PE-CAM-1), defined by the CD31 monoclonal antibody (MoAb), was initially described as a cell-cell adhesion molecule mediating both homotypic and heterotypic adhesion. In this report, we show that enriched CD34+ human hematopoietic progenitor cell populations, containing early myeloid, erythroid, and multipotential progenitor cells, are CD31+. Analyses of CD34+ cell lines representing early myeloid, multipotential, and pre- pre-B-lymphoid progenitors indicate that precursors of both myeloid and B-lymphoid cells express PECAM-1 at high levels. Three-color flow- cytometric analyses also show that normal human bone marrow CD31+ CD34+ subsets coexpress myeloid (CD33) or B-lymphoid (CD19, CD10) markers. Except for the monocytic cell line, U937, all CD34- cell lines tested, which represent more mature stages of the myeloid, erythroid, and lymphoid lineages, expressed substantially lower or negligible levels of PECAM-1. Western blotting studies indicated that the CD31 MoAb, JC/70A, detected molecules in the 120- to 140-kD molecular weight range on the monocytic CD34- CD33+ CD31+ cell line, U937; on the CD34+ CD31+ CD33+ CD19- multipotential/lymphomyeloid precursor cell lines, KG1 and KG1B; on the CD34+ CD31+ CD19+ CD10+ CD33- precursor pre-pre-B-cell line, MIK-ALL; and on a CD34(+)-enriched precursor cell population from normal human bone marrow. A single molecular weight species was generally observed with enriched membrane preparations, whereas two PECAM-1 molecules were present in whole-cell lysates of cell lines and the CD34+ bone marrow cell subset. Preliminary studies show that a proportion of the PECAM-1 molecules on the lymphomyeloid/multipotential progenitor cell line, KG1, and on the monocytic cell line, U937, binds to heparin-sepharose. A soluble form of PECAM-1 also binds heparin- sepharose. The high level of expression of PECAM-1 on CD34+ cells suggests that this glycoprotein may function as a heterotypic adhesion molecule, possibly mediating multipotential, myeloid, and early-B- lymphoid precursor cell interactions with stromal cells and extracellular matrix molecules via heparan sulfate proteoglycans. It may also act as a homotypic adhesion molecule by interacting with PECAM- 1 on bone marrow stromal macrophage-like cells and endothelial cells or on endothelial cells during stem/progenitor cell migration. Thus, this molecule has the potential importance of directing both lineage commitment and trafficking of early hematopoietic progenitor cells.     

Unique patterns of surface receptors, cytokine secretion, and immune functions distinguish T cells in the bone marrow from those in the periphery: impact on allogeneic bone marrow transplantation

The "conventional" NK1.1(-) T cells from mouse blood and marrow were compared with regard to surface receptors, cytokine secretion, and function. Most blood NK1.1(-) CD4(+) and CD8(+) T cells expressed the naive CD44(int/lo)CD62L(hi)CD45RB(hi) T-cell phenotype typical of those in the peripheral lymphoid tissues. In contrast, most marrow NK1.1(-) CD4(+) and CD8(+) T cells expressed an unusual CD44(hi)CD62L(hi)CD45RB(hi) phenotype. The blood NK1.1(-) CD4(+) T cells had a naive T-helper cytokine profile and a potent capacity to induce lethal graft versus host (GVH) disease in a C57BL/6 donor to a BALB/c host bone marrow transplantation model. In contrast, the marrow NK1.1(-) CD4(+) T cells had a Th0 cytokine profile and failed to induce lethal GVH disease, even at 20-fold higher numbers than those from the blood. NK1.1(-) CD8(+) T cells from the blood but not the marrow induced lethal GVH disease. Nevertheless, the marrow NK1.1(-) CD8(+) T cells induced potent antitumor activity that was augmented by marrow NK1.1(-) CD4(+) T cells and facilitated hematopoietic progenitor engraftment. The inability of marrow CD4(+) and CD8(+) T cells to induce GVH disease was associated with their inability to expand in the blood and gut of allogeneic recipients. Because neither the purified marrow CD4(+) or CD8(+) T cells induced GVH disease, their unique features are desirable for inclusion in allogeneic bone marrow or hematopoietic progenitor transplants.     

Serotonin 5-HT2B receptors are required for bone-marrow contribution to pulmonary arterial hypertension

Pulmonary arterial hypertension (PAH) is a progressive disease characterized by lung endothelial dysfunction and vascular remodeling. Recently, bone marrow progenitor cells have been localized to PAH lungs, raising the question of their role in disease progression. Independently, serotonin (5-HT) and its receptors have been identified as contributors to the PAH pathogenesis. We hypothesized that 1 of these receptors, 5-HT(2B), is involved in bone marrow stem cell mobilization that participates in the development of PAH and pulmonary vascular remodeling. A first study revealed expression of 5-HT(2B) receptors by circulating c-kit(+) precursor cells, whereas mice lacking 5-HT(2B) receptors showed alterations in platelets and monocyte-macrophage numbers, and in myeloid lineages of bone marrow. Strikingly, mice with restricted expression of 5-HT(2B) receptors in bone marrow cells developed hypoxia or monocrotaline-induced increase in pulmonary pressure and vascular remodeling, whereas restricted elimination of 5-HT(2B) receptors on bone marrow cells confers a complete resistance. Moreover, ex vivo culture of human CD34(+) or mice c-kit(+) progenitor cells in the presence of a 5-HT(2B) receptor antagonist resulted in altered myeloid differentiation potential. Thus, we demonstrate that activation of 5-HT(2B) receptors on bone marrow lineage progenitors is critical for the development of PAH.     

Prolonged ex vivo culture of human bone marrow mesenchymal stem cells influences their supportive activity toward NOD/SCID-repopulating cells and committed progenitor cells of B lymphoid and myeloid lineages

Background

Bone marrow mesenchymal stem cells support proliferation and differentiation of hematopoietic progenitor cells in vitro. Since these cells constitute a rare subset of bone marrow cells, mesenchymal stem cell preparations for clinical purposes require a preparative step of ex vivo multiplication. The aim of our study was to analyze the influence of culture duration on mesenchymal stem cell supportive activity.

Design and Methods

Mesenchymal stem cells were expanded for up to ten passages. These cells and CD34+ cells were seeded in cytokine-free co-cultures after which the phenotype, clonogenic capacity and in vivo repopulating activity of harvested hematopoietic cells were assessed.

Results

Early passage mesenchymal stem cells supported hematopoietic progenitor cell expansion and differentiation toward both B lymphoid and myeloid lineages. Late passage mesenchymal stem cells did not support hematopoietic progenitor cell and myeloid cell outgrowth but maintained B-cell supportive ability. In vitro maintenance of NOD/SCID mouse repopulating cells cultured for 1 week in contact with mesenchymal stem cells was effective until the fourth passage of the mesenchymal cells and declined thereafter. The levels of engraftment of CD34⁺ cells in NOD/SCID mice was higher when these cells were co-injected with early passage mesenchymal stem cells; however mesenchymal cells expanded beyond nine passages were ineffective in promoting CD34⁺ cell engraftment. Non-contact cultures indicated that mesenchymal stem cell supportive activity involved diffusible factors. Among these, interleukins 6 and 8 contributed to the supportive activity of early passage mesenchymal stem cells but not to those of late passage cells. The phenotype, as well as fat, bone and cartilage differentiation capacity, of mesenchymal stem cells did not change during their culture.

Conclusions

Extended culture of mesenchymal stem cells alters the ability of these cells to support hematopoietic progenitor cells without causing concomitant changes in their phenotype or differentiation capacity.     

Delivery of Flt3 ligand (Flt3L) using a poloxamer-based formulation increases biological activity in mice

Fms-like tyrosine kinase (Flt3L) is a potent stimulator of hematopoietic progenitor cell (HPC) expansion and mobilization; however, this requires 7-10 days of administration. We investigated whether sustained delivery of Flt3L using a poloxamer-based matrix (PG) could accelerate and/or improve the hematopoietic activity of Flt3L in mice. A single injection of PG-Flt3L stimulated significantly more rapid and greater HPC mobilization to the spleen and peripheral blood than the daily injection of Flt3L formulated in saline. Pharmacokinetic analysis demonstrated that the formulation of Flt3L in PG prolonged its elimination (Tbeta) half-life (2.3-fold) and increased its bioavailability (>two fold) and the time to maximum serum concentration (T(max)) (2.7-fold). Further, coadministration of G-CSF and PG-Flt3L allowed lower doses of Flt3L to be active, with significantly greater hematopoietic and mobilization activity, compared to the same total dose of G-CSF, Flt3L or G-CSF and Flt3L formulated in saline. These data demonstrate that formulation of Flt3L in PG significantly accelerates and increases HPC expansion and mobilization. The observation of increased bioactivity by PG-Flt3L in rodents suggests the potential for improved clinical efficacy of Flt3L by reducing the time required for HPC mobilization.     

Interleukin 3 directly stimulates both megakaryocyte progenitor cells and immature megakaryocytes.

A subset of stem cell antigen (sca-1)-positive mouse megakaryocyte progenitors was identified that correlates with other primitive precursors in bone marrow. The responsive bone marrow cells were obtained by depleting the marrow of cells bearing defined lineage markers (neutrophils, macrophages, and lymphoid cells) and enriched for primitive myeloid progenitor cells with high proliferative potential, selecting for cells expressing sca-1. The sca-1-positive megakaryocyte progenitors formed colonies in the presence of interleukin 3 (IL-3) alone. Immature megakaryocytes depleted of mature megakaryocytes and of cells expressing myeloid and lymphoid lineage markers were also responsive to IL-3. These data indicate that in the presence of high doses of IL-3, accessory cells are not obligatory for growth factor stimulation of megakaryocytopoiesis in vitro.     

Human bone marrow stromal cells simultaneously support B and T/NK lineage development from human haematopoietic progenitors: a principal role for flt3 ligand in lymphopoiesis

The regulation of human early lymphopoiesis remains unclear. B- and T-lineage cells cannot develop simultaneously with conventional stromal cultures. Here we show that telomerized human bone marrow stromal cells supported simultaneous generation of CD19(+) CD34(lo/-) CD10(+) cyCD79a(+) CD20(+/-) VpreB(-) pro-B cells and CD7(+) CD34(+) CD45RA(+) CD56(-) cyCD3(-) early T/Natural Killer (NK) cell precursors from human haematopoietic progenitors, and the generation of both lymphoid precursors was promoted by flt3 ligand (flt3L). On the other hand, stem cell factor or thrombopoietin had little or no effect when used alone. However, both acted synergistically with flt3L to augment the generation of both lymphoid precursors. Characteristics of these lymphoid precursors were evaluated by gene expression profiles, rearrangements of IgH genes, or replating assays. Similar findings were observed with primary human bone marrow stromal cells. Notably, these two lymphoid-lineage precursors were generated without direct contact with stromal cells, indicating that early B and T/NK development can occur, at least in part, by stromal cell-derived humoral factors. In serum-free cultures, flt3L elicited similar effects and appeared particularly important for B cell development. The findings of this study identified the potential of human bone marrow stromal cells to support human early B and T lymphopoiesis and a principal role for flt3L during early lymphopoiesis.     

Disruption of the estrogen receptor beta gene in mice causes myeloproliferative disease resembling chronic myeloid leukemia with lymphoid blast crisis

Proliferation of pluripotent, bone marrow stem cells, which develop to lymphoid and myeloid progenitors, is negatively regulated by estrogen. Although in estrogen deficiency and in estrogen receptor knockout mice there is significant alteration in bone marrow hematopoiesis, the effects of aging on estrogen receptor deficiencies in mice have not been investigated yet. In this study we show that by 1.5 years of age, estrogen receptor beta knockout (ERbeta-/-) mice develop pronounced splenomegaly that is much more severe in females than in males. Further characterization of these mice revealed myelogenous hyperplasia in bone marrow, an increase in the number of granulocytes and B lymphocytes in blood, lymphadenopathy, and infiltration of leukocytes in the liver and lung. Analysis by flow cytometry of the bone marrow cells revealed that the percentage and total number of Gr-1hi/Mac-1hi-positive granulocytes were increased by 15-30% and 100%, respectively. The numbers of B cells in the bone marrow and spleen were significantly higher in ERbeta-/- mice than in WT littermates. Some of the ERbeta-/- mice also had a severe lymphoproliferative phenotype. Thus the absence of ERbeta results in a myeloproliferative disease resembling human chronic myeloid leukemia with lymphoid blast crisis. Our results indicate a previously unknown role for ERbeta in regulating the differentiation of pluripotent hematopoietic progenitor cells and suggest that the ERbeta-/- mouse is a potential model for myeloid and lymphoid leukemia. Furthermore, we suggest that ERbeta agonists might have clinical value in the treatment of leukemia.     

Vav promoter-tTA conditional transgene expression system for hematopoietic cells drives high level expression in developing B and T cells

OBJECTIVE: We previously showed that Vav promoter-tetracycline transactivator (Vav-tTA)-driven tetracycline-regulated element (TRE)-NRAS(V12) expression resulted in mastocytosis development in mice. To investigate which hematopoietic cells express TRE-driven transgenes when combined with Vav-tTA, we assayed hematopoietic cells, including bone marrow-derived mast cells (BMMC) and CD34-positive hematopoietic progenitor cells (HPC) as well as myeloid and lymphoid lineages. To determine if suppression of NRAS(V12) expression early in life would delay mastocytosis we treated developing and juvenile mice with doxycycline (Dox). MATERIALS AND METHODS: Vav-tTA-driven luciferase expression was assayed by live mouse imaging and relative light unit measurement before or after treating Vav-tTA and TRE-luciferase (TRE-Luc) cotransgenic mice with Dox. Magnetic cell sorting and fluorescence-activating cell sorting methods were used to sort hematopoietic cells. To suppress TRE-mediated luciferase or NRAS(V12) expression in Vav-tTA cotransgenic mice, we added Dox to the drinking water. RESULTS: B cells in the bone marrow and T cells in the thymus expressed Vav-tTA-driven luciferase at much higher levels than in myeloid cells, BMMC, and CD34-positive HPC, which showed relatively low levels. Dox treatment completely eliminated the luciferase expression from all hematopoietic cells. Repression of TRE-NRAS(V12) expression early in life was sufficient to increase the latency of mastocytosis development. CONCLUSION: The Vav-tTA transgenic line will be very useful for conditional transgene expression in developing B and T cells. Vav-tTA-driven NRAS(V12) expression is sufficient for mastocytosis development, but not for myeloid leukemia. Lymphoid cells are resistant to NRAS(V12) transformation despite high level of expression.