Human CD34+ HLA-DR- bone marrow cells contain progenitor cells capable of self-renewal, multilineage differentiation, and long-term in vitro hematopoiesis.

Human bone marrow cells expressing CD34 but not HLA-DR were isolated by immunofluorescence flow cytometric cell sorting. These cells contained a hematopoietic cell (CFU-B1) capable of producing, in an in vitro semisolid culture system, blast-cell-containing colonies, which possessed the capacity for self-renewal and commitment to multipotential differentiation. In addition, CD34+ HLA-DR- marrow cells contained primitive megakaryocyte progenitor cells, the burst-forming unit-megakaryocyte (BFU-MK). A subset of CD34+ HLA-DR- marrow cells lacking the expression of CD15 and CD71 was obtained by flow cytometric cell sorting and was capable of sustaining in vitro hematopoiesis in suspension culture for up to 8 weeks in the absence of a preestablished adherent marrow cell layer. The combination of IL-3 + IL-1 alpha and IL-3 + IL-6 sustained proliferation of these cells for 8 weeks, induced maximal cellular expansion, and increased the numbers of assayable progenitor cells. These studies demonstrate that human CD34+ HLA-DR- marrow cells and their subsets contain primitive multipotential hematopoietic cells capable of self-renewal and of differentiation into multiple hematopoietic lineages.     

MERP1: a mammalian ependymin-related protein gene differentially expressed in hematopoietic cells

We have utilized differential display polymerase chain reaction to investigate the gene expression of hematopoietic progenitor cells from adult bone marrow and umbilical cord blood. A differentially expressed gene was identified in CD34+ hematopoietic progenitor cells, with low expression in CD34- cells. We have obtained the full coding sequence of this gene which we designated human mammalian ependymin-related protein 1 (MERP1). Expression of MERP1 was found in a variety of normal human tissues, and is 4- and 10-fold higher in adult bone marrow and umbilical cord blood CD34+ cells, respectively, compared to CD34- cells. Additionally, MERP1 expression in a hematopoietic stem cell enriched population was down-regulated with proliferation and differentiation. Conceptual translation of the MERP1 open reading frame reveals significant homology to two families of glycoprotein calcium-dependant cell adhesion molecules: ependymins and protocadherins.     

Identification of a hierarchy of multipotent hematopoietic progenitors in human cord blood

Mouse hematopoiesis is initiated by long-term hematopoietic stem cells (HSC) that differentiate into a series of multipotent progenitors that exhibit progressively diminished self-renewal ability. In human hematopoiesis, populations enriched for HSC activity have been identified, as have downstream lineage-committed progenitors, but multipotent progenitor activity has not been uniquely isolated. Previous reports indicate that human HSC are enriched in Lin-CD34+CD38- cord blood and bone marrow and express CD90. We demonstrate that the Lin-CD34+CD38- fraction of cord blood and bone marrow can be subdivided into three subpopulations: CD90+CD45RA-, CD90-CD45RA-, and CD90-CD45RA+. Utilizing in vivo transplantation studies and complementary in vitro assays, we demonstrate that the Lin-CD34+CD38-CD90+CD45RA- cord blood fraction contains HSC and isolate this activity to as few as 10 purified cells. Furthermore, we report the first prospective isolation of a population of candidate human multipotent progenitors, Lin-CD34+CD38-CD90-CD45RA- cord blood.     

Simultaneous use of rhodamine 123, phycoerythrin, Texas red, and allophycocyanin for the isolation of human hematopoietic progenitor cells

The supravital, mitochondrial specific dye Rhodamine 123 (R123) was used in conjunction with three monoclonal antibodies to isolate a population of human bone marrow (BM) cells enriched for hematopoietic progenitor cells. BM cells stained with phycoerythrin-HLA-DR, Texas red-CD34, allophycocyanin-CD15, and R123 were fractionated using four-color immunofluorescence cell sorting. Cells expressing CD34 but not HLA-DR and CD15 (CD34+ HLA-DR- CD15-) were subdivided according to their reactivity with R123 into quiescent, R123 dull (R+) or cycling, R123 bright (R++) subpopulations. Morphological analysis and hematopoietic progenitor cell assays indicated that CD34+ HLA-DR- CD15- R+ cells contained larger numbers of blast cells and colony forming units than CD34+ HLA-DR- CD15- R++ cells. The flow cytometer settings used to accommodate the detection of the R123 fluorescence in combination with that of three other fluorochromes are described.     

Human T-cell leukemia virus infection of human hematopoietic progenitor cells: maintenance of virus infection during differentiation in vitro and in vivo.

Human T-cell leukemia virus type I (HTLV-1) is the etiologic agent of adult T-cell leukemia and lymphoma and HTLV-1-associated myelopathy-tropical spastic paraparesis. We examined whether HTLV could productively infect human hematopoietic progenitor cells. CD34+ cells were enriched from human fetal liver cells and cocultivated with cell lines transformed with HTLV-1 and -2. HTLV-1 infection was established in between 10 and >95% of the enriched CD34+ cell population, as demonstrated by quantitative PCR analysis. HTLV-1 p19 Gag expression was also detected in infected hematopoietic progenitor cells. HTLV-1-infected hematopoietic progenitor cells were cultured in semisolid medium permissive for the development of erythbroid (BFU-E), myeloid (CFU-GM), and primitive progenitor (CFU-GEMM, HPP-CFC, or CFU-A) colonies. HTLV-1 sequences were detected in colonies of all hematopoietic lineages; furthermore, the ratio of HTLV genomes to the number of human cells in each infected colony was 1:1, consistent with each colony arising from a single infected hematopoietic progenitor cell. Severe combined immunodeficient mice engrafted with human fetal thymus and liver tissues (SCID-hu) develop a conjoint organ which supports human thymocyte differentiation and maturation. Inoculation of SCID-hu mice with HTLV-1-infected T cells or enriched populations of CD34+ cells established viral infection of thymocytes 4 to 6 weeks postreconstitution. Thymocytes from two mice with the greatest HTLV-1 proviral burdens showed increased expression of the CD25 marker and the interleukin 2 receptor alpha chain and perturbation of CD4+ and CD8+ thymocyte subset distribution profiles. Hematopoietic progenitor cells and thymuses may be targets for HTLV infection in humans, and these events may play a role in the pathogenesis associated with infection.     

人造血干／祖细胞多态性的研究：：Ⅺ.人正常骨髓CD34^＋造血细胞周 …

DNA, RNA and PRO comprise the bulk of macromolecules in cells, which have been proven to play an important role in regulating cell cycle transverse capacity, cell division, growth, and size. Simultaneous analysis of these moieties could provide more comprehensive and accurate information on cell cycle kinetics. In this study, DNA, RNA and PRO contents related to cell cycle kinetics in CD34+ hematopoietic cells of human bone marrow were measured to understanding the cell cycle kinetic features in CD34+ hematopoietic cells. For this reason, CIMS-100 immunomagnetic isolator, a novel isolation system, was used to enrich efficiently CD34+ hematopoietic cells from human bone marrow. The purity of enriched CD34+ hematopoietic cells determined by both FACS and APAAP staining is ranging from 90%-95%. Cellular DNA, RNA and PRO were stained with fluorochromes propidium iodide, pyronin Y and fluorescein isothiocyante respectively The fluorescence intensities reflecting the DNA, RNA and PRO content of individual cell were analyzed in FACS can by different excitation wavelengthes. DNA, RNA and PRO contents in CD34+ hematopoietic cells were far lower than these of bone marrow mononuclear cells, only being 34 +/- 3% (DNA), 48 +/- 21% (RNA) and 62 +/- 14% (PRO) of BMMNCs respectively. Collectively, these data combined with previous results from both ours and others indicated that CD34+ hematopoietic cells are indeed an unique cell population, not only in reconstitute of hematopoietic and immunological functions, but also in cell cycle kinetics. This is, to our knowledge, the first detailed report on the analysis of DNA, RNA and PRO contents related to cell cycle kinetics in CD34+ hematopoietic cells. And the results provide more direct evidence that the majority of CD34+ hematopoietic cells are in resting state.     

Eltrombopag, a thrombopoietin receptor agonist, enhances human umbilical cord blood hematopoietic stem/primitive progenitor cell expansion and promotes multi-lineage hematopoiesis

Umbilical cord blood (UCB) transplantation has emerged as a promising therapy, but it is challenged by scarcity of stem cells. Eltrombopag is a non-peptide, thrombopoietin (TPO) receptor agonist, which selectively activates c-Mpl in humans and chimpanzees. We investigated eltrombopag's effects on human UCB hematopoietic stem cell (HSC) and hematopoietic progenitor cell (HPC) expansion, and its effects on hematopoiesis in vivo. Eltrombopag selectively augmented the expansion of human CD45+, CD34+, and CD41+ cells in bone marrow compartment without effects on mouse bone marrow cells in the NOD/SCID mice xenotransplant model. Consequently, eltrombopag increased peripheral human platelets and white blood cells. We further examined effects in the STAT and AKT signaling pathways in serum-free cultures. Eltrombopag expanded human CD34+ CD38-, CD34+, and CD41+ cells. Both eltrombopag and recombinant human TPO (rhTPO) induced phosphorylation of STAT5 of CD34+ CD41-, CD34- CD41+, and CD34- CD41- cells. rhTPO preferentially induced pSTAT3, pAKT, and more pSTAT5 in CD34- C41+ cells, while eltrombopag had no effects on pSTAT3. In conclusion, eltrombopag enhanced expansion of HSCs/HPCs of human UCB in vivo and in vitro, and promoted multi-lineage hematopoiesis through the expansion of bone marrow HSCs/HPCs of human UCB in vivo. Eltrombopag differed somewhat from rhTPO in the signal transduction pathways by favoring earlier HSC/HPC populations.     

Single-cell image analysis to assess ABC-transporter-mediated efflux in highly purified hematopoietic progenitors

BACKGROUND: Normal and malignant hematopoietic stem cells are characterized by their capacity to actively extrude fluorescent dyes. The contribution of different ATP-binding cassette (ABC) transporters to this phenomenon is largely unknown due to the small stem cell numbers limiting the use of standard methods to assess functional efflux. METHODS: We used epifluorescence microscopy (EFM) in combination with single-cell image analysis to study ABC-transporter-mediated efflux in highly purified, viable, CD34+CD38- cells sorted on an adhesive biolayer. P-glycoprotein and multidrug-resistant protein (MRP)-mediated efflux were quantitated using fluorescent substrates (rhodamine-123 and calcein acetoxymethyl ester [calcein-AM]) and specific inhibitors (verapamil and probenecid, respectively). RESULTS: The feasibility, sensitivity, and reproducibility of rhodamine-123 efflux quantitation using single-cell EFM was shown in cell lines and compared with standard flow cytometric assessment. P-glycoprotein-mediated transport was higher in CD34+CD38- cells than in more differentiated progenitors (mean efflux index = 2.24 +/- 0.35 and 1.14 +/- 0.11, respectively; P = 0.01). P-glycoprotein-mediated transport was the main determinant of the rhodamine "dull" phenotype of these cells. In addition, significant MRP-mediated efflux was demonstrated in CD34+CD38- and CD38+ cells (mean efflux index = 1.42 +/- 0.19 and 1.28 +/- 0.18, respectively). CONCLUSION: The described method is a valuable tool for assessing ABC-transporter-mediated efflux in highly purified single cells. Both P-glycoprotein and MRP-mediated efflux are present in human CD34+CD38- hematopoietic stem cells.     

Leukemic Stem Cell Frequency: A Strong Biomarker for Clinical Outcome in Acute Myeloid Leukemia

Introduction

Treatment failure in acute myeloid leukemia is probably caused by the presence of leukemia initiating cells, also referred to as leukemic stem cells, at diagnosis and their persistence after therapy. Specific identification of leukemia stem cells and their discrimination from normal hematopoietic stem cells would greatly contribute to risk stratification and could predict possible relapses.

Results

For identification of leukemic stem cells, we developed flow cytometric methods using leukemic stem cell associated markers and newly-defined (light scatter) aberrancies. The nature of the putative leukemic stem cells and normal hematopoietic stem cells, present in the same patient''s bone marrow, was demonstrated in eight patients by the presence or absence of molecular aberrancies and/or leukemic engraftment in NOD-SCID IL-2Rγ-/- mice. At diagnosis (n = 88), the frequency of the thus defined neoplastic part of CD34+CD38- putative stem cell compartment had a strong prognostic impact, while the neoplastic parts of the CD34+CD38+ and CD34- putative stem cell compartments had no prognostic impact at all. After different courses of therapy, higher percentages of neoplastic CD34+CD38- cells in complete remission strongly correlated with shorter patient survival (n = 91). Moreover, combining neoplastic CD34+CD38- frequencies with frequencies of minimal residual disease cells (n = 91), which reflect the total neoplastic burden, revealed four patient groups with different survival.

Conclusion and Perspective

Discrimination between putative leukemia stem cells and normal hematopoietic stem cells in this large-scale study allowed to demonstrate the clinical importance of putative CD34+CD38- leukemia stem cells in AML. Moreover, it offers new opportunities for the development of therapies directed against leukemia stem cells, that would spare normal hematopoietic stem cells, and, moreover, enables in vivo and ex vivo screening for potential efficacy and toxicity of new therapies.     

CD41 expression defines the onset of primitive and definitive hematopoiesis in the murine embryo

The platelet glycoprotein IIb (alpha(IIb); CD41) constitutes the alpha subunit of a highly expressed platelet surface integrin protein. We demonstrate that CD41 serves as the earliest marker of primitive erythroid progenitor cells in the embryonic day 7 (E7.0) yolk sac and high-level expression identifies essentially all E8.25 yolk sac definitive hematopoietic progenitors. Some definitive hematopoietic progenitor cells in the fetal liver and bone marrow also express CD41. Hematopoietic stem cell competitive repopulating ability is present in CD41(dim) and CD41(lo/-) cells isolated from bone marrow and fetal liver cells, however, activity is enriched in the CD41(lo/-) cells. CD41(bright) yolk sac definitive progenitor cells co-express CD61 and bind fibrinogen, demonstrating receptor function. Thus, CD41 expression marks the onset of primitive and definitive hematopoiesis in the murine embryo and persists as a marker of some stem and progenitor cell populations in the fetal liver and adult marrow, suggesting novel roles for this integrin.     

Modelling of ex vivo expansion/maintenance of hematopoietic stem cells

In this study, we described the modelling of the expansion/maintenance of human hematopoietic stem/progenitor cells from adult human bone marrow. CD 34(+)-enriched cell populations from bone marrow were cultured in the presence and absence of human stroma in serum-free media containing bFGF, SCF, LIF and Flt-3 ligand for several days. The cells in the culture were analysed for expansion and phenotype by flow cytometry. Although significant expansion of bone marrow cultures occurred in the presence and absence of human stroma, the results of expansion were effectively better in the presence of a stromal layer. In both situations the phenotypic analysis demonstrated a great expansion of CD 34(+)38(-) cells. The differentiative potential of bone marrow CD 34(+) cells co-cultured with human stroma was primarily shifted towards the myeloid lineage with the presence of CD 15 and CD 33.     

Cell surface sialylation and ecto-sialyltransferase activity of human CD34 progenitors from peripheral blood and bone marrow

Surface expressed negatively charged sialoglycans contribute to the regulation of adhesive cellular interactions and are thus involved in the growth and differentiaton of hematopoietic progenitor cells. In particular, the cell surface sialylation state may govern the liberation of CD34+ hematopoietic precursors from bone marrow stroma cells and extracellular matrix. In order to assess the overall surface sialylation of live human CD34+ hematopoietic precursor cells, we applied a previously described flow cytometric enzyme assay. Cells with and without sialidase pretreatment were incubated in the presence of fluorescent CMP-sialic acid and exogenous ST6GalI. Thus sialylation of surface-expressed lactosamine residues was analysed. We demonstrated that surface lactosamines of CD34+ precursors derived from bone marrow and peripheral blood are over 95% sialylated, predominantly in 2-6 linkage. These results are in accordance with flow cytometric analysis of surface lectin staining. Sialic acid specific lectins MAA and SNA were strongly bound whereas SBA, VVA, and PNA became reactive only after sialidase pretreatment. CD34+ leukemia cell lines TF1 and KG1a also showed a high degree of surface sialylation, whereas cell line KG1 expressed to the largest extent free lactosamines. In these cell lines, 2-6 and 2-3 sialylated residues were present in equal amounts. In a variation of the flow cytometric enzyme assay, live cells were incubated without exogenous STGal I to measure the activity of endogenous ecto-sialyltransferase. Ecto sialyltransferase activity was observed in all CD34+ cells which was able to resialylate major surface glycoproteins such as HLA Class I, CD45, CD43, and CD34. The ecto-sialyltransferase may serve to maintain or increase surface sialylation rapidly without de novo synthesis. Published in 2004.     

Comparative analysis of differential expression of sialic acids and adhesion molecules on mononuclear cells of bone marrow and peripheral blood in childhood acute lymphoblastic leukaemia at diagnosis and clinical remission

Childhood acute lymphoblastic leukaemia (ALL) is characterized by the neoplasm of immature haematopoietic precursor cells (HPCs). We report significant differences between the expression of sialoglycoproteins and adhesion molecules on mononuclear cells (MNCs) of bone marrow (BM) and peripheral blood (PB) from individual children at diagnosis of the disease. Lymphoblasts in PB predominantly expressed 9-O-acetylated sialoglycoproteins (Neu5,9Ac2-GPs), sialic acid, alpha2-3 linked sialic acid, L- and P-selectins and vascular cell adhesion molecule -1 (VCAM-1) on their surface compared to BM, as determined with selective lectins and monoclonal antibodies (mAbs) by flow cytometric analysis. CD34+CD38+ cells present either in diagnostic PB or BM always showed enhanced expression of both alpha2-3 and alpha2-6 linked sialic acids, Neu5,9Ac2-GPs, L- and P-selectins and VCAM-1, compared to CD34+CD38- population, as confirmed by higher mean fluorescence intensity (MFI). Expression of ICAM-1 was reverse. However, MFI of Neu5,9Ac2-GPs was always higher both in CD34+CD38+ and CD34+CD38- population in PB compared to BM. Diverse trend of these cell surface macromolecules was observed during clinical remission. This is the first comparative study between PB and BM, where significant differential distribution of sialylated macromolecules and adhesion molecules was observed. Hence, supervising these cell surface macromolecules at various stages of treatment might help in minimal residual disease detection, identifying mobilization factor(s) and in isolation of normal HPCs for autologous BM transplantation.     

Inhibition of human bone marrow lymphoid progenitor colonies by antibodies to VLA integrins.

Studies in animal models suggest that the integrin adhesion protein VLA-4 may play an important role in lymphopoiesis. The relationship between cell adhesion and lymphopoiesis in humans has been difficult to study because of the relative rarity and stringent in vitro growth requirements of lymphoid progenitors from normal adult human bone marrow. To determine the functional significance of VLA-4-mediated adhesion in human lymphopoiesis, we developed a culture system in which a bone marrow-derived adherent layer supports the formation of colonies of terminal deoxynucleotidyl transferase (TdT)-positive lymphoid precursor cells from normal adult human bone marrow. Limiting dilution studies were consistent with clonal origin of these colonies. CFU-TdT were enriched in the CD34+ bone marrow fraction, consistent with CD34 expression by other hematopoietic progenitors. CD34 expression and lack of lineage-specific markers in a significant proportion of the TdT+ colony cells suggest that the TdT+ CFU may represent an uncommitted lymphoid progenitor cell. Development of TdT+ colonies required direct contact with the adherent layer and was significantly inhibited by specific anti-VLA-4 alpha chain antibody, suggesting a functional role for the previously reported VLA-4-dependent adhesion of human B cell precursors to bone marrow-derived fibroblasts.     

Testosterone influences renal electrolyte excretion in SHR/y and WKY males

Background

Aldehyde dehydrogenase (ALDH) is a cytosolic enzyme highly expressed in hematopoietic precursors from cord blood and granulocyte-colony stimulating factor mobilized peripheral blood, as well as in bone marrow from patients with acute myeloblastic leukemia. As regards human normal bone marrow, detailed characterization of ALDH⁺ cells has been addressed by one single study (Gentry et al, 2007). The goal of our work was to provide new information about the dissection of normal bone marrow progenitor cells based upon the simultaneous detection by flow cytometry of ALDH and early hematopoietic antigens, with particular attention to the expression of ALDH on erythroid precursors. To this aim, we used three kinds of approach: i) multidimensional analytical flow cytometry, detecting ALDH and early hematopoietic antigens in normal bone marrow; ii) fluorescence activated cell sorting of distinct subpopulations of progenitor cells, followed by in vitro induction of erythroid differentiation; iii) detection of ALDH⁺ cellular subsets in bone marrow from pure red cell aplasia patients.

Results

In normal bone marrow, we identified three populations of cells, namely ALDH⁺CD34⁺, ALDH^-CD34⁺ and ALDH⁺CD34^- (median percentages were 0.52, 0.53 and 0.57, respectively). As compared to ALDH^-CD34⁺ cells, ALDH⁺CD34⁺ cells expressed the phenotypic profile of primitive hematopoietic progenitor cells, with brighter expression of CD117 and CD133, accompanied by lower display of CD38 and CD45RA. Of interest, ALDH⁺CD34^- population disclosed a straightforward erythroid commitment, on the basis of three orders of evidences. First of all, ALDH⁺CD34^- cells showed a CD71^bright, CD105⁺, CD45^- phenotype. Secondly, induction of differentiation experiments evidenced a clear-cut expression of glycophorin A (CD235a). Finally, ALDH⁺CD34^- precursors were not detectable in patients with pure red cell aplasia (PRCA).

Conclusion

Our study, comparing surface antigen expression of ALDH⁺/CD34⁺, ALDH^-/CD34⁺ and ALDH⁺/CD34^- progenitor cell subsets in human bone marrow, clearly indicated that ALDH⁺CD34^- cells are mainly committed towards erythropoiesis. To the best of our knowledge this finding is new and could be useful for basic studies about normal erythropoietic differentiation as well as for enabling the employment of ALDH as a red cell marker in polychromatic flow cytometry characterization of bone marrow from patients with aplastic anemia and myelodysplasia.     

Cellular determinants affecting the rate of cytokine in cultures of human hematopoietic cells

The present study was undertaken to define parameters that may limit the cytokine-mediated expansion of primitive hematopoietic cells in stirred suspension cultures of normal human marrow cells. In a first series of experiments, parallel measurements of the rate and extent of progenitor expansion and cytokine depletion from the medium were made for such cultures in which the cells were exposed to different cytokine concentrations. Supplementation of the medium with 2 ng/mL of interleukin-3 (IL-3), IL-6 and IL-11 plus 10 ng/mL of Flt-3 ligand (FL) and Steel factor (SF) allowed a 45-fold expansion of directly clonogenic cell (CFC) numbers within 2 weeks along with a 2.5-fold expansion of their precursors, detectable as longterm culture-initiating cells (LTC-IC). The addition of 5-fold higher levels of these cytokines enhanced the 2 week output of both CFC and LTC-IC numbers (to 66-fold and 9-fold above input respectively). However, this was also associated with an increase in the individual average rates of depletion of immunoreactive IL-3, SF and FL. As a result, even biweekly addition of fresh medium supplemented with the highest concentrations of cytokines tested failed to prevent a continuing decline in their levels relative to the input medium levels. A similar dependence of the IL-3 depletion rate on its extracellular concentration was demonstrable in suspension cultures of Mo7e cells, an IL-3-dependent human leukemic cell line.Additional experiments with various highly purified marrow cell fractions showed that the rate of cytokine depletion varied according to the type of responding cell as well as the specific cytokine. CD34(+)CD38(-) cells exhibited the greatest average cell-specific cytokine depletion rates (35-fold higher than unseparated bone marrow cells). These findings establish new principles that will be important for the optimization of hematopoietic cell bioreactors. In addition, they suggest that cytokine depletion may provide a novel feedback control mechanism in vivo which would contribute to the control of primitive hematopoietic cell proliferation and differentiation. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 54: 58-66, 1997.     

Ex vivo expansion of primitive hematopoietic cells for cellular therapies: An overview

Sources of hematopoietic cells for bone marrow transplantation are limited by the supply of compatible donors, the possibility of viral infection, and autologous (patient) marrow that is depleted from prior chemo- or radiotherapy or has cancerous involvement. Anex vivo system to amplify hematopoietic progenitor cells could increase the number of patients eligible for autologous transplant, allow use of cord blood hematopoietic cells to repopulate an adult, reduce the amount of bone marrow and/or mobilized peripheral blood stem and progenitor cells required for transplantation, and reduce the time to white cell and platelet engraftment. The cloning of hematopoietic growth factors and the identification of appropriate conditions has enabled the development of successfulex vivo hematopoietic cell cultures. Purification systems based on the CD34 marker (which is expressed by the most primitive hematopoietic cells) have proven an essential tool for research and clinical applications. Present methods for hematopoietic cultures (HC) on stromal (i.e. accessory cells that support hematopoiesis) layers in flasks lack a well-controlled growth environment. Several bioreactor configurations have been investigated, and a first generation of reactors and cultures has reached the clinical trial stage. Our research suggests that perfusion conditions improve substantially the performance of hematopoietic reactors. We have designed and tested a perfusion bioreactor system which is suitable for the culture of non-adherent cells (without stromal cells) and readily scaleable for clinical therapies. Eliminating the stromal layer eliminates the need for a stromal cell donor, reduces culture time, and simplifies the culture system. In addition, we have compared the expansion characteristics of both mononuclear and CD34⁺ cells, since the latter are frequently assumed to give a superior performance for likely transplantation therapies.Abbreviations BFU0-E burst forming unit-erythroid - BM bone marrow - CB cord blood - CFU-C colony forming unit-culture - CFU-E colony forming unit-erythroid - CFU-F colony forming unit-fibroblast - CFU-GEMM colony forming unit-granulocyte, erythroid, macrophage, megakaryocyte - CFU-GM colony forming unit-granulocyte, macrophage - CFU-Mix colony forming unit-mixed (also known as CFU-GEMM) - CML chronic myeloid leukemia - CSF colony stimulating factor - DMSO dimethyl sulfoxide - ECM extracellular matrix - EPO erythropoietin - FL fetal liver - HC hematopoietic culture - LTBMC long-term bone marrow culture - LTC-IC long-term culture initiating cell - LTHC long-term hematopoietic culture - MNC mononuclear cells - PB peripheral blood     

Flow cytometric enumeration of CD34+ hematopoietic stem and progenitor cells in leukapheresis product and bone marrow for clinical transplantation: a comparison of three methods

Flow cytometric enumeration of CD34+ hematopoietic stem and progenitor cells (HSCs) is widely used for evaluation of graft adequacy of peripheral blood and bone marrow stem cell grafts. In the present study, we review and compare the major counting techniques of stem and progenitor cells. The methods are: the Milan/Mullhouse protocol, two-platform ISHAGE (International Society of Hematotherapy and Graft Engineering) and single-platform ISHAGE analysis system. According to the Milan/Mulhouse protocol, HSCs are identified by CD34 antibody staining and easy gating strategy. The ISHAGE guidelines for detection of CD34+ cells are based on a four-parameter flow cytometry method (CD34PE/CD45PerCP staining, side and forward angle light scatter) thus employing multiparameter gating strategy. With two-platform ISHAGE protocol, an absolute CD34+ count is generated by incorporating the leukocyte count from an automated hematology analyser. The single-platform ISHAGE method to determine the absolute CD34+ count directly from a flow cytometer includes the use of Trucount tubes (Becton Dickinson) with a known number of fluorescent beads. CD34+ cells were quantified in mobilized peripheral blood, collected by leukapheresis, and bone marrow from 42 samples from patients with hematological malignancies. The differences against the means display low disagreement between the Milan/Mulhouse and ISHAGE protocols, with discrepancies of up to 2.5% (two-platform ISHAGE)--2.6% (single-platform ISHAGE) in enumeration of CD34+ cells in leukapheresis product and 4.8% (two-platform ISHAGE)--4.9% (single-platform ISHAGE) in bone marrow. Our results show high correlation among all three methods. Since the three protocols are compatible, choosing the most convenient in terms of costs, simplicity and compliance with clinical results appears to be a logical consequence.     

Signaling through toll-like receptor 7/8 induces the differentiation of human bone marrow CD34+ progenitor cells along the myeloid lineage

Toll-like receptors (TLRs) play a key role in pathogen recognition and regulation of the innate and adaptive immune responses. Although TLR expression and signaling have been investigated in blood cells, it is currently unknown whether their bone marrow ancestors express TLRs and respond to their ligands. Here we found that TLRs (e.g. TLR4, TLR7 and TLR8) were expressed by freshly isolated human bone marrow (BM) hematopoietic CD34+ progenitor cells. Incubation of these primitive cells with TLR ligands such as immunostimulatory small interfering RNAs and R848, a specific ligand for TLR7/8, induced cytokine production (e.g. IL1-beta, IL6, IL8, TNF-alpha, GM-CSF). Moreover, TLR7/8 signaling induced the differentiation of BM CD34+ progenitors into cells with the morphology of macrophages and monocytic dendritic precursors characterized by the expression of CD13, CD14 and/or CD11c markers. By contrast, R848 ligand did not induce the expression of glycophorin A, an early marker for erythropoiesis. Collectively, the data indicate for the first time that human BM CD34+ progenitor cells constitutively express functional TLR7/TLR8, whose ligation can induce leukopoiesis without the addition of any exogenous cytokines. Thus, TLR signaling may regulate BM cell development in humans.