Culture Of Hematopoietic Progenitor Cells Research Articles

Induction of deubiquitinating enzyme USP50 during erythropoiesis and its potential role in the regulation of Ku70 stability

Anemia is a very common blood disorder that affects the lives of billions of people worldwide. Anemia is caused by the loss of blood, increased destruction of red blood cells...

Covalent immobilization of stem cell factor and stromal derived factor 1α for in vitro culture of hematopoietic progenitor cells

Hematopoietic stem cells (HSCs) are currently utilized in the treatment of blood diseases, but widespread application of HSC therapeutics has been hindered by the limited availability of HSCs. With a better understanding of the HSC microenvironment and the ability to precisely recapitulate its components, we may be able to gain control of HSC behavior. In this work we developed a novel, biomimetic PEG hydrogel material as a substrate for this purpose and tested its potential with an anchorage-independent hematopoietic cell line, 32D clone 3 cells. We immobilized a fibronectin-derived adhesive peptide sequence, RGDS; a cytokine critical in HSC self-renewal, stem cell factor (SCF); and a chemokine important in HSC homing and lodging, stromal derived factor 1α (SDF1α), onto the surfaces of poly(ethylene glycol) (PEG) hydrogels. To evaluate the system’s capabilities, we observed the effects of the biomolecules on 32D cell adhesion and morphology. We demonstrated that the incorporation of RGDS onto the surfaces promotes 32D cell adhesion in a dose-dependent fashion. We also observed an additive response in adhesion on surfaces with RGDS in combination with either SCF or SDF1α. In addition, the average cell area increased and circularity decreased on gel surfaces containing immobilized SCF or SDF1α, indicating enhanced cell spreading. By recapitulating aspects of the HSC microenvironment using a PEG hydrogel scaffold, we have shown the ability to control the adhesion and spreading of the 32D cells and demonstrated the potential of the system for the culture of primary hematopoietic cell populations.

Characterizing functional heterogeneity in hematopoietic progenitor cell cultures: a combined experimental and modeling approach

Cultures of primary HSCs and hematopoietic cell lines are often characterized by heterogeneous proliferation rates, a non-negligible level of background differentiation and cell death and the maintenance of self-renewal ability within a small subpopulation of cells only. The multipotent murine progenitor cell line FDCP-Mix demonstrates these features during continuous passaging in high IL-3. The mechanisms by which a small fraction of self-renewing cells is maintained in a continuously expanding, intrinsically heterogeneous population are unknown, but are likely to be relevant to similar homeostatic processes in vivo.

Erythrocyte enrichment in hematopoietic progenitor cell cultures based on magnetic susceptibility of the hemoglobin.

Using novel media formulations, it has been demonstrated that human placenta and umbilical cord blood-derived CD34+ cells can be expanded and differentiated into erythroid cells with high efficiency. However, obtaining mature and functional erythrocytes from the immature cell cultures with high purity and in an efficient manner remains a significant challenge. A distinguishing feature of a reticulocyte and maturing erythrocyte is the increasing concentration of hemoglobin and decreasing cell volume that results in increased cell magnetophoretic mobility (MM) when exposed to high magnetic fields and gradients, under anoxic conditions. Taking advantage of these initial observations, we studied a noninvasive (label-free) magnetic separation and analysis process to enrich and identify cultured functional erythrocytes. In addition to the magnetic cell separation and cell motion analysis in the magnetic field, the cell cultures were characterized for cell sedimentation rate, cell volume distributions using differential interference microscopy, immunophenotyping (glycophorin A), hemoglobin concentration and shear-induced deformability (elongation index, EI, by ektacytometry) to test for mature erythrocyte attributes. A commercial, packed column high-gradient magnetic separator (HGMS) was used for magnetic separation. The magnetically enriched fraction comprised 80% of the maturing cells (predominantly reticulocytes) that showed near 70% overlap of EI with the reference cord blood-derived RBC and over 50% overlap with the adult donor RBCs. The results demonstrate feasibility of label-free magnetic enrichment of erythrocyte fraction of CD34+ progenitor-derived cultures based on the presence of paramagnetic hemoglobin in the maturing erythrocytes.

Establishment of an aging model of Sca-1+ hematopoietic stem cell and studies on its relative biological mechanisms

The objective of this study is to establish the aging model of murine hematopoietic stem cell (HSC) ex vitro and investigate its relative biological mechanism, aimed to build the foundation for searching the methods to delaying HSC aging. Sca-1(+)HSC were isolated and purified from murine bone marrow mono-nucleated cell by magnetic-activated cell sorting. The purity of separated cells was analyzed by flow cytometry (FCM) and the expression of Sca-1 was detected by immunofluorescence. Sca-1(+)HSC induced aging by tert-butylhydroperoxide (t-BHP, final concentration of 100μmol/L) for 6h to establish the murine HSC aging model in vitro. Biological characteristics of aging HSC were evaluated by mixed hematopoietic progenitor cell culture in vitro, cell cycle assay and senescence-associated β-galactosidase (SA-β-gal) cytochemical staining. Telomere length and telomerase activity were detected by southern blotting and telomere repeat amplification protocol-polymerase chain reaction (TRAP-PCR) augmentation. The expressions of p16(INK4a), P19(Arf), P53, P21(Cip1/Waf1) mRNA were detected by reverse transcription (RT)-PCR. The purity of separated Sca-1(+) HSC was 87.2% and the survival of Sca-1(+) HSC was 96~99%. After 6h cocultured with 100μmol/Lt-BHP, the ability of aging Sca-1(+) HSC to form mixed hematopoietic progenitor colony, self-renewal and multi-differentiation were decreased significantly. The number of aging Sca-1(+) HSC entered G1 phase of the cell cycle and the percentage of positive cells expressed SA-β-gal increased significantly. The telomere length shortened and the telomerase activity decreased. The expression of p16(INK4a), p19(Arf), p53, P21(Cip1/Waf1) mRNA increased. The t-BHP can induce Sca-1(+) HSC senescence in vitro. The signal transduction pathways of p16(INK4a)-retinoblastoma and P19(Arf)-Mdm2-P53-P21(Cip1/Waf1) may play key roles in the Sca-1(+) HSC senescence induced by t-BHP.

Maintenance and expansion of hematopoietic stem/progenitor cells in biomimetic osteoblast niche

In this study, we employed bio-derived bone scaffold and composited with the marrow mesenchymal stem cell induced into osteoblast to replicate a "biomimetic niche." The CD34(+) cells or mononuclear cells (MNC) from umbilical cord blood were cultured for 2-5weeks in the biomimetic niche (3D system) was compared with conventional two dimensional cultures (2D system) without adding cytokine supplement. After 2weeks in culture, the CD34(+) cells from umbilical cord blood in the 3D system increased 3.3-4.8 folds when compared with the initial CD34(+) cells. CD34(+)/CD38(-) cells accounted for 82-90% of CD34(+) cells. After 5weeks, CD34(+)/CD38(-) cells in the 3D system increased when compared with initial (1.3±0.3×10(3) vs. 1.0±0.5×10(4), p<0.05), but were decreased in the 2D system (1.3±0.3×10(3) vs. 2.5±0.7×10(2), p<0.05). The CFU progenitors were produced more in the 3D system than in the 2D system (4.6-9.3 folds vs. 1.0-1.5 folds) after 2weeks in culture, and the colony distribution in the 3D system manifested higher percentage of BFU-E and CFU-GEMM, but in the 2D system was mainly CFU-GM. The LTC-ICs in the 3D system showed 5.2-7.2 folds increase over input at 2weeks in culture, and maintain the immaturation of hematopoietic progenitor cells (HPCs) over 5weeks. In conclusion, this new 3D hematopoietic progenitor cell culture system is the first to utilize natural cancellous bone as scaffold with osteoblasts as supporting cells; it is mimicry of natural bone marrow HSC niche. Our primary work has demonstrated it could maintain and expand HSC/HPC in vitro.

Controle de esterilidade de produtos de células progenitoras hematopoéticas do sangue periférico

A taxa de contaminação microbiana dos produtos de células progenitoras hematopoéticas do sangue periférico é baixa. Neste estudo pesquisou-se a prevalência de hemoculturas positivas em células progenitoras hematopoéticas do sangue periférico (CPHSP) no Serviço de Hemoterapia do Hospital de Clínicas de Porto Alegre. Do total de 618 coletas realizadas no período de 2000 a 2007, 26 (4,2%) apresentaram contaminação por bactérias. O Staphylococcus coagulase-negativo foi predominantemente isolado nas hemoculturas. A antibioticoterapia pré e pós-infusão foi estabelecida de acordo com o microorganismo e seu antibiograma, sendo que, em cinco das doze infusões contaminadas realizadas, não foram administrados antimicrobianos profilaticamente. Episódios febris foram observados em sete pacientes (58%), enquanto cinco (42%) não apresentaram febre. Das doze infusões contaminadas realizadas, seis (50%) apresentaram hemocultura pós-descongelamento positivas, enquanto as restantes (50%) foram negativas. Isto se deve às propriedades bactericidas do DMSO, de células fagocitose-ativas e de temperaturas muito baixas atingidas na criopreservação. Autores têm relatado sucesso neste procedimento após a infusão desses produtos contaminados com o mínimo de consequências clínicas.

NFI-A directs the fate of hematopoietic progenitors to the erythroid or granulocytic lineage and controls β-globin and G-CSF receptor expression

AbstractIt is generally conceded that selective combinations of transcription factors determine hematopoietic lineage commitment and differentiation. Here we show that in normal human hematopoiesis the transcription factor nuclear factor I-A (NFI-A) exhibits a marked lineage-specific expression pattern: it is upmodulated in the erythroid (E) lineage while fully suppressed in the granulopoietic (G) series. In unilineage E culture of hematopoietic progenitor cells (HPCs), NFI-A overexpression or knockdown accelerates or blocks erythropoiesis, respectively: notably, NFI-A overexpression restores E differentiation in the presence of low or minimal erythropoietin stimulus. Conversely, NFI-A ectopic expression in unilineage G culture induces a sharp inhibition of granulopoiesis. Finally, in bilineage E + G culture, NFI-A overexpression or suppression drives HPCs into the E or G differentiation pathways, respectively. These NFI-A actions are mediated, at least in part, by a dual and opposite transcriptional action: direct binding and activation or repression of the promoters of the β-globin and G-CSF receptor gene, respectively. Altogether, these results indicate that, in early hematopoiesis, the NFI-A expression level acts as a novel factor channeling HPCs into either the E or G lineage.

Copper Modulates the Differentiation of Mouse Hematopoietic Progenitor Cells in Culture

Copper chelation has been shown to favor the expansion of human hematopoietic stem/progenitor cells in vitro. To further understand the effects of copper modulation on defined subsets of stem cells versus progenitor cells, we extended the studies in a mouse system. We isolated mouse hematopoietic stem cells (HSCs) or hematopoietic progenitor cells (HPCs) and cultured them with or without the copper chelator tetraethylenepentamine (TEPA) or CuCl(2). Cytokine-stimulated HPC cultures treated with TEPA for 7 days generated about two to three times more total and erythroid colony-forming cells (CFCs) compared to control cultures. In contrast, CuCl(2) treatment decreased the CFC numbers. Similar results were seen with HSC after 14, but not 7, days of culture. Transplant studies showed that HPCs cultured for 7 days in TEPA had about twofold higher short-term erythroid repopulation potential compared to control cultures, while CuCl(2) decreased the erythroid potential of cultured HPCs compared to control cultures. HSCs cultured with TEPA for 7 days did not exhibit significantly higher repopulation potential in either leukocyte or erythrocyte lineages compared to control cultures in short-term or long-term assays. Based on JC-1 staining, the mitochondrial membrane potential of HPCs cultured with TEPA was lower relative to control cultures. Our data suggest that decreasing the cellular copper content with TEPA results in preferential expansion or maintenance of HPC that are biased for erythroid differentiation in vivo, but does not enhance the maintenance of HSC activity in culture.

The TLR1/2 agonist PAM3CSK4 instructs commitment of human hematopoietic stem cells to a myeloid cell fate

Toll-like receptors (TLRs) constitute a family of nonpolymorphic receptors that are devoted to pathogen recognition. In this work, we have explored the impact of TLR ligands (TLR-L) on human hematopoietic stem cells (HSCs) and hematopoietic progenitor cells (HPCs). We show that HSCs and HPCs have a comparable pattern of expression of TLR transcripts characterized by the predominance of TLR1, -2, -3, -4 and -6. In long-term cultures of HSCs, HPCs and stromal cells, most TLR-L profoundly inhibited B-cell development while preserving or enhancing the production of myeloid cells. In short-term cultures, the TLR1/2 ligand PAM(3)CSK(4) induced a large proportion of HPCs to express markers of the myelomonocytic lineage. PAM(3)CSK(4) induced only marginal expression of myeloid lineage markers on HSCs but promoted their myeloid commitment as revealed by their acquisition of the phenotype of multi- and bipotential myeloid progenitors and by upregulation of the transcription factors PU.1, C/EBPalpha and GATA-1. Our results suggest that TLR agonists can bias the lineage commitment of human HSCs and shift the differentiation of lineage-committed progenitors to favor myelopoiesis at the expense of lymphoid B-cell development.

Negative regulation of activated α2 integrins during thrombopoiesis

AbstractCirculating platelets exhibit rapid signaling and adhesive responses to collagen that facilitate hemostasis at sites of vessel injury. Because platelets are anuclear, their collagen receptors must be expressed by megakaryocytes, platelet precursors that arise in the collagen-rich environment of the bone marrow. Whether and how megakaryocytes regulate collagen adhesion during their development in the bone marrow are unknown. We find that surface expression of activated, but not wild-type, α2 integrins in hematopoietic cells in vivo results in the generation of platelets that lack surface α2 receptors. Culture of hematopoietic progenitor cells ex vivo reveals that surface levels of activated, but not wild-type, α2 integrin receptors are rapidly down-regulated during cell growth on collagen but reach wild-type levels when cells are grown in the absence of collagen. Progenitor cells that express activated α2 integrins are normally distributed in the bone marrow in vivo and exhibit normal migration across a collagen-coated membrane ex vivo. This migration is accompanied by rapid down-regulation of activated surface integrins. These studies identify ligand-dependent removal of activated α2 receptors from the cell surface as a mechanism by which integrin function can be negatively regulated in hematopoietic cells during migration between the adhesive environment of the bone marrow and the nonadhesive environment of the circulating blood.

Association Of Hematopoietic Stem Cell Product Culture And Clinical Infection In Patients Receiving Myeloablative Chemotherapy With Autologous Hematopoietic Stem Cell Reinfusion

Purpose: The purpose of this descriptive retrospective QA project is to examine the association between hematopoietic progenitor cell (HPC) product cultures at the time of reinfusion and the subsequent development of infection. Methods: Using a descriptive, retrospective design, data were collected from the records of 68 patients who received autologous peripheral blood HPC reinfusions after undergoing myeloablative chemotherapy at a single institution. Consistent with the oncology service QA program, all of the products were tested for microbial contamination at the time of stem cell processing. At the time of reinfusion, a sample from each thawed product was sent to the microbiology lab. Following the HPC reinfusion, blood cultures were sent on patients at the time of their first fever and at least once daily until they were afebrile and cultures were negative. Blood cultures were sent from each lumen of the central venous catheter. Peripheral blood cultures were not routinely drawn. Results: Between March 2002 and September 2008, 68 patients received 148 HPC reinfusions. All of the reinfused HPC products were negative for bacterial contamination at the time of processing. Nineteen of the reinfused HPC cultures, which were reinfused into 13 patients, were positive. Two of these patients developed positive blood cultures. Patient #1 had HPC product culture positive for Bacillus species and Staphhylococcus epidermis. Blood cultures were positive only for S. epidermis on day +15. Patient #2 had HPC product cultures positive for Streptococcus oralis. Blood cultures were positive for Streptococcus mitis on day +4 and S. epidermis on day +6. Conclusions: There does not appear to be a strong correlation between positive product cultures and the subsequent development of positve blood cultures. Microbial testing at the time of HPC reinfusion may not be necessary.

Ex vivo expansion of hematopoietic progenitor cells is associated with downregulation of 4 integrin- and CXCR4-mediated engraftment in NOD/SCID 2-microglobulin-null mice

Several studies indicate that ex vivo cytokine-supported expansion induces defective hematopoietic stem cell engraftment. We investigated the role of alpha4 integrin, alpha5 integrin and CXCR4 in engraftment of unmanipulated and cytokine-treated human cord blood CD34(+) cells. Uncultured or expanded CD34(+) cells were infused in NOD/SCID-beta(2)microglobulin-null mice. The function of alpha4, and alpha5 integrins and CXCR4 was assessed by incubating cells with specific neutralizing antibodies, prior to transplant. The activation state of alpha4 integrin was further tested by adhesion and migration assays. Neutralization of either alpha4 integrin or CXCR4 abolished engraftment of uncultured CD34(+) cells at 6 week spost-transplant, while alpha5 integrin neutralization had no significant effect. However, after short-term ex vivo culture, blocking alpha4 integrin or CXCR4 did not affect repopulating activity whereas neutralization of alpha5 integrin inhibited engraftment. Using soluble vascular cell adhesion molecule-1 binding assays, we observed that alpha4 integrin affinity in fresh CD34(+) cells was low and susceptible to stimulation while in cultured CD34(+) cells, it was high and insensitive to further activation. In addition, stromal cell-derived factor-1 stimulated migration across vascular cell adhesion molecule-1 in fresh CD34(+) cells but not in cultured CD34(+) cells. Our data show that ex vivo culture of hematopoietic progenitor cells is associated with downregulation of both alpha4 integrin- and CXCR4-mediated engraftment. Further investigations suggest that this is caused by supraphysiological increase of alpha4 integrin affinity, which impairs directional migration across vascular cell adhesion molecule-1 in response to stromal cell-derived factor-1. Such changes may underlie the engraftment defect of cytokine-stimulated CD34(+) cells.

Photodynamic treatment with mono‐phenyl‐tri‐(N‐methyl‐4‐pyridyl)‐porphyrin for pathogen inactivation in cord blood stem cell products

Hematopoietic stem cell transplants and culture of hematopoietic progenitor cells require pathogen-free conditions. The application of a method of pathogen inactivation in red blood cells using photodynamic treatment (PDT) was investigated for the decontamination of cord blood stem cell (CBSC) products. CBSC products, spiked with Gram-positive and Gram-negative bacteria, were treated with PDT using mono-phenyl-tri-(N-methyl-4-pyridyl)-porphyrin (Tri-P(4)) and red light. After PDT, in vitro and in vivo evaluation of the CBSC functions were performed. PDT of CBSC products resulted in the inactivation of the bacteria, with Staphylococcus aureus being the most resistant. Complete decontamination was achieved when CBSC products were contaminated with low titers of bacteria. PDT had no effect on white blood cell viability, the ex vivo expansion potential of the progenitor cells, and their capacity to differentiate to various hematopoietic cell lineages. However, PDT reduced the engraftment of human CBSCs in NOD/SCID mice, particularly affecting the B-cell lineage engraftment. Pathogen inactivation of CBSC with Tri-P(4)-mediated PDT is feasible at contamination level up to 10 to 20 colony-forming units per mL and can be considered when ex vivo expansion culture is anticipated. However, this treatment is not recommended for transplantation purposes at this time. Further investigations may elucidate why engraftment is diminished.

Viability does not necessarily reflect the hematopoietic progenitor cell potency of a cord blood unit: results of an interlaboratory exercise

Clinical transplant outcome with umbilical cord blood (UCB) as source of hematopoietic progenitor cells (HPCs) is, among other factors, determined by the total number of viable nucleated cells and/or CD34+ cells in the unit. Quantitative and qualitative losses by processing and cryopreservation and by thawing and washing before transfusion may occur, however. Another reason for a discrepancy between the number of cells in the unit released by the cord blood bank and found in the transplant center may be technical differences in cell counting methods between the two sites. With the collaborative group for Biomedical Excellence for Safer Transfusion (BEST), an interlaboratory exercise was conducted among nine sites for thawed UCB variables: total nucleated cells, CD34+ cells, viability, and HPC cultures. Three frozen UCB samples were shipped, with instructions for thawing, counting, and HPC plating. Unexpectedly samples arrived at all nine receiving centers without detectable hematopoietic progenitor colony-forming cells. Nevertheless, wide interlaboratory ranges for viability were obtained. The proportion of viable cells was found higher with manual methods, but all viability assays used in the study overestimated functional progenitor cells. The results underscore the complexity of evaluation of frozen-thawed cord blood cells and the need for standardization of assessment.

Excessive Isolated Erythrocytosis Linked to Induction of SCL/Tal1 and Erythropoietin Receptor Expression.

The JAK2 V617F mutation has been detected in more than 90% polycythemia vera patients and is associated with increased erythropoietin (EPO) sensitivity or EPO independence of erythroid progenitor cells during early erythropoiesis. To determine if other molecular lesions give rise to increased erythrocytosis and realizing that the JAK2 V617F mutation may not be the initiating event in polycythemia vera, we identified an individual with isolated, excessive erythrocytosis who showed erythroid precursor hypersensitivity to EPO; no mutations on sequencing genes for erythropoietin receptor (EPO-R), VHL, or HIF-1 IRE; normal serum EPO level; normal hemoglobin function and p50; normal cardiac, hepatic and pulmonary function; no JAK2 V617F mutation and no EPO independent BFU-E. While it was not certain that this individual's erythrocytosis was lifelong, other relatives had increased hemoglobin levels. The cause of this individual's increased erythropoiesis was investigated using EPO-stimulated cultures of hematopoietic progenitor cells isolated from peripheral blood. As with normal controls, cell numbers were not increased after 12 days of incubation with low levels of EPO (<0.1 U/ml). With 1U/ml of EPO, cell proliferation was similar to control early in erythropoiesis but increased markedly after 8 days so that by day 12 cell numbers were 3-fold greater than in control cultures with 95% benzidine positive cells compared with 76% for control and 82–87% for JAK2 V617F positive erythroid progenitor cells. Analyses of transcription factor expression revealed that induction of GATA-1 and down regulation of GATA-2 were similar to control. However, SCL/Tal1 and EKLF markedly increased beginning at day 8 to 10 and continued to rise during late erythropoiesis in contrast to control and JAK2 V617F positive cultures in which SCL/Tal1 and EKLF peaked at day 10 and decreased with late erythroid differentiation. Since increased beta-globin expression is concomitant with induction of EKLF, the rise in EKLF may account for the marked increase in benzidine positive cells from our subject. Moreover, expression of EPO-R followed the continuing rise in SCL/Tal1, increasing by 3.5 fold at day 12 compared with control cultures. This high EPO-R expression is consistent with the dramatic increase in cell proliferation during late erythropoiesis. Using forced expression of SCL/Tal1, reporter gene assay and gel mobility shift analysis in erythroid cells, we determined that SCL/Tal1 is able to bind to E-boxes located 3′ of the EPO-R proximal promoter and to activate transcription. Although EPO stimulation of erythroid progenitor cells activates EPO-R, we found that forced expression of EPO-R in primary erythroid progenitor cells in the presence of EPO increases expression of GATA-1 as well as SCL/Tal1 and EKLF, providing further evidence that specific increase in SCL/Tal1 but not GATA-1 must precede induction of EPO-R in this case of excessive erythrocytosis. These data demonstrate a link between high level induction of SCL/Tal1 expression and increased erythrocytosis and suggest a mechanism that contributes to increased erythropoietin sensitivity during late erythropoiesis.

Hypoxia alters progression of the erythroid program

Hypoxia can induce erythropoiesis through regulated increase of erythropoietin (Epo) production. We investigated the direct influence of oxygen tension (pO(2)) in the physiologic range (2-8%) on erythroid progenitor cell differentiation using cultures of adult human hematopoietic progenitor cells exposed to decreasing (20% to 2%) pO(2) and independent of variation in Epo levels. Decreases in hemoglobin (Hb)-containing cells were observed at the end of the culture period with decreasing pO(2). This is due, in part, to a reduction in cell growth and, at 2% O(2), a marked increase in cell toxicity. Analysis of the kinetics of cell differentiation showed an increase in the proportion of cells with glycophorin-A expression and Hb accumulation at physiologic pO(2). Cells were characterized by an early induction of gamma-globin expression and a delay and reduction in peak levels of beta-globin expression. Overall, fetal Hb and gamma-globin expression were increased at physiologic pO(2), but these increases were reduced at 2% O(2) as cultures become cytotoxic. At reduced pO(2), induction of Epo-receptor (Epo-R) by Epo was decreased and delayed, analogous to the delay in beta-globin induction. The oxygen-dependent reduction of Epo-R can account for the associated cytotoxicity at 2% O(2). Epo induction of erythroid transcription factors, EKLF, GATA-1, and SCL/Tal-1, was also delayed and decreased at reduced pO(2), consistent with lower levels of Epo-R and resultant Epo signaling. These changes in Epo-R and globin gene expression raise the possibility that the early increase of gamma-globin is a consequence of reduced Epo signaling and a delay in induction of erythroid transcription factors.

MicroRNAs 17-5p/20a/106a Function as a Master Gene Complex Controlling Monocytopoiesis through AML1 Targeting.

MicroRNAs (miRs) are a novel regulatory class of non-coding, single-stranded RNAs of ~ 22 nucleotides, identified in plants and animals. MiRs repress protein expression at post-transcriptional level, mostly through base pairing to the 3′ untranslated region (UTR) of the target mRNA, thus leading to its degradation and/or reduced translation. MiRs have been shown to control basic biological functions, such as cell proliferation and differentiation. However, little is known on their role in hematopoiesis. Furthermore, in diverse types of cancer miRs may function as “oncomirs”.We investigated the role of miR-17-5p, -20a and -106a, located in the miR-17-5p-92 and miR-106a-92 clusters respectively, in monocytic-macrophage (Mo) differentiation-maturation. In unilineage Mo culture generated by cord blood (CB) hematopoietic progenitor cells (HPC), these miRs are gradually and markedly downmodulated, whereas AML1 is upmodulated at protein but not at mRNA level. The acute myeloid leukaemia-1 protein (AML1, also known as Runt-related transcription factor 1, Runx1) is the DNA-binding subunit of the hematopoietic transcription factor CBF, which plays a pivotal role in Mo differentiation-maturation. Luciferase assay shows that miR-17/20/106 bind the AML1 mRNA 3′UTR, implying that during monocytopoiesis their decline unblocks AML1 translation. Mo culture transfection with miR-17/20/106 stimulates blast cell proliferation and inhibits differentiation-maturation: this is coupled with AML1 translation blockade, leading in turn to inhibition of M-CSF receptor (M-CSFR) expression. Equivalent results were observed in Mo clonogenic culture of HPCs. Knockdown of endogenous miR-17/20/106 by anti-miR oligonucleotide transfection causes opposite effects. Altoghether, our studies indicate that miR-17/20/106 downmodulation during monocytopoiesis “unblocks” the translation of AML1, which in turn transactivates the M-CSFR, thereby allowing Mo differentiation and maturation. We hence propose that these miRs function as a master gene complex underlying the monocytopoiesis gene program, at least in part through the regulation of two pivotal proteins, AML1 and M-CSFR.This study also sheds light on other relevant aspects:the regulatory action of miR-17/20/106 action on AML1 mRNA is restricted to the Mo lineage, implying a role for lineage-specific factor(s) to modulate the interaction of these miRs with their mRNA target;the subtle complexity of mechanisms controlling the transcription/processing of the miR-17-5p-92 cluster, implied by the differential expression patterns of the miRs comprised in the 17-5p-92 polycistron in Mo culture;the leukemogenesis mediated by AML1 fusion proteins.

457. The Benefits of CH-296 during Ex Vivo Gene Transfer Go beyond the Ability to Co-Localize the Retroviral Particle and Target Cell

Successful gene therapy depends on at least two parameters: introduction of the corrective gene into primitive hematopoietic progenitor cells (HPC) and maintenance of the long-term engraftment potential of these ex vivo manipulated progenitors post-transplantation. The use of the recombinant fibronectin fragment, CH-296, increases retroviral gene transfer by co-localizing the retroviral particle and the target cell which expresses VLA-4 and VLA-5 integrins. In a xenotransplantation model, our lab previously demonstrated the maintenance of long-term reconstituting hematopoietic progenitors during ex vivo gene transfer either on a pre-established irradiated bone marrow stromal monolayer or CH-296-coated plates. In contrast, HPC cultured on BSA-coated plates in the presence of IL-3, IL-6, and SCF maintained short- but not long-term engraftment potential. Here, we compared the molecular changes that occur in bone marrow and cord blood HPC cultured on CH-296 vs. BSA-coated plates in the presence of cytokines. Bone marrow HPC were cultured in the presence of IL-3, IL-6, and SCF for 48 hours. Subcellular fractionation followed by immunoblotting showed an induction of p21Cip1 protein in the cytoplasmic compartment of HPC cultured on BSA-coated plates. Since p21Cip1 is upregulated during monocytic differentiation, we hypothesized that lack of integrin engagement in the presence of cytokines might induce the loss of HPC primitive characteristics. Confirming our hypothesis, we observed a loss of c-myb, CD34, and GATA-2 expression in HPC maintained in suspension. In addition, we observed changes in MAPK phosphorylation and localization in HPC cultured on fibronectin, as compared to BSA-coated plates. Extrapolating the same hypothesis to cordblood derived HPCs, we observed consistently higher levels of reactive oxygen species (ROS) in cordblood HPC on BSA-coated plates, as compared to cells on CH-296. Aside from regulating cell differentiation, high levels of ROS have been shown to induce apoptosis. Consistent with this, we documented lower levels of phosphorylated cytoplasmic AKT in HPC on BSA-coated plates, as compared to cells on CH-296 plates. Based on these preliminary observations, we hypothesize that the suppression of differentiation markers such as p21Cip1 and apoptotic inducers such as intracellular reactive oxygen species, the maintenance of transcription factors such as GATA-2 and c-Myb, and the enhanced phosphorylation of the pro-survival regulator AKT in HPCs during ex vivo culture on CH-296 may all contribute to the ability of these cells to achieve long term engraftment following transplantation into sublethally irradiated immunodeficient mice. We therefore propose that inclusion of CH-296 during ex vivo culture of HPC may be important even during non-retroviral mediated gene transfer in order to maintain HPC engraftment potential.

IL-10 Is a Novel Ligand for Hematopoietic Stem Cell Self-Renewal.

IL-10 has been known to play a major role in modulating inflammatory and immune response. However, the anemia sign in targeted disruption of IL-10 (IL-10KO) and supportive effects of IL-10 during in-vitro culture of hematopoietic progenitor cells implicated potential role of IL-10 for hematopoietic function.To delineate the physiological significance of IL-10 for normal hematopoiesis, first, we undertook study to examine the various hematopoietic compartments in IL-10 KO mice in comparison to wild type (WT) counterparts.In the phenotypic analysis, the bone marrow cells (BMC) of IL-10 KO mice showed modest hypocellularity including TER119+ cells, but no difference were observed in the assay for in-vitro colony forming cells (CFC) (3 Exp.).In contrast, the number of more primitive hematopoietic cells as defined by long term culture (LTC-IC) was 2-fold lower in the BMCs of IL-10 KO mice compared to WT, and further decrease (7-fold) were observed in those IL-10 KO mice that had enterocolitis (2 Exp). Similarly, when equivalent numbers of BMCs from IL-10 KO or WT were transplanted into irradiated congeneic recipient (Pep3b-Ly5.1), IL-10 KO BMC showed 2-fold lower level of engraftment (88% vs 44%) than WT over the span of post-transplantation 3 to 12 weeks (2 exp, n=7 ea,). In the limiting dilution analysis to measure the HSC contents, the frequency of competitive repopulating unit (CRU) in the IL-10 KO BMC was also two fold lower than WT (1/2164 vs. 1/5931, respectively) with further decreasse in CRU frequency being observed for those from IL-10 KO with enterocolitis (< 1/17380), suggesting that IL-10 KO mice has decreased content of hematopoietic stem cell (HSC).To exclude possible indirect biological effects in IL-10 KO mice, and see if IL-10 have direct effect on quantity of HSCs, normal 5-FU prestimulated BMCs were cultured for 5 days on stromal cells that had been retrovirally transduced with IL-10 or control vector (MIG) along with TPO, SCF and FL. When the cells were transplanted into the recipients, cells cultured in the IL-10 secreting stroma exhibited significantly higher level engraftment compared to those cultured in the control stroma (23% vs 54% for PB, 16% vs 49% for BM engraftment, respectively, n=4). Secondary transplantation of these primary recipients at post-transplantation 9 month showed that the BM cells grown in IL-10 secreting stroma had 663 donor-derived CRUs, while those transplanted with control group had 215 donor-derived CRUs, a 3-fold higher CRU contents in the presence of IL-10. In further studies to see if IL-10 could be a direct ligand for primitive HSCs, first, expression of IL-10 receptor on those cells were confirmed by RT-PCR. Next, purified HSCs (c-kit+Sca-1+Lin-) without accessory cells were cultured in the stroma-free condition with or without exogeneous addition of IL-10 for 5 days, and transplanted into recipients in limiting dilution. The result showed that CRU frequency of cells cultured with IL-10 was 3-fold higher than those cultured in the absence of IL-10 (1/525 vs. 1/1465), thus suggesting that IL-10 is a direct ligand for HSC self-renewal. These results may implicate significance of IL-10 for pathogenesis and treatment of autoimmune diseases by stem cell transplantation as well as for improved ex-vivo expansion of HSCs.