Loss Of Hematopoietic Progenitor Cells Research Articles

Arginine methyltransferase PRMT5 is essential for sustaining normal adult hematopoiesis.

Epigenetic regulators play critical roles in normal hematopoiesis, and the activity of these enzymes is frequently altered in hematopoietic cancers. The major type II protein arginine methyltransferase PRMT5 catalyzes the formation of symmetric dimethyl arginine and has been implicated in various cellular processes, including pluripotency and tumorigenesis. Here, we generated Prmt5 conditional KO mice to evaluate the contribution of PRMT5 to adult hematopoiesis. Loss of PRMT5 triggered an initial but transient expansion of hematopoietic stem cells (HSCs); however, Prmt5 deletion resulted in a concurrent loss of hematopoietic progenitor cells (HPCs), leading to fatal BM aplasia. PRMT5-specific effects on hematopoiesis were cell intrinsic and depended on PRMT5 methyltransferase activity. We found that PRMT5-deficient hematopoietic stem and progenitor cells exhibited severely impaired cytokine signaling as well as upregulation of p53 and expression of its downstream targets. Together, our results demonstrate that PRMT5 plays distinct roles in the behavior of HSCs compared with HPCs and is essential for the maintenance of adult hematopoietic cells.

GABP transcription factor is required for development of chronic myelogenous leukemia via its control of PRKD2

Hematopoietic stem cells (HSCs) are the source of all blood lineages, and HSCs must balance quiescence, self-renewal, and differentiation to meet lifelong needs for blood cell development. Transformation of HSCs by the breakpoint cluster region-ABL tyrosine kinase (BCR-ABL) oncogene causes chronic myelogenous leukemia (CML). The E-twenty six (ets) transcription factor GA binding protein (GABP) is a tetrameric transcription factor complex that contains GABPα and GABPβ proteins. Deletion in bone marrow of Gabpa, the gene that encodes the DNA-binding component, caused cell cycle arrest in HSCs and profound loss of hematopoietic progenitor cells. Loss of Gabpα prevented development of CML, although mice continued to generate BCR-ABL-expressing Gabpα-null cells for months that were serially transplantable and contributed to all lineages in secondary recipients. A bioinformatic screen identified the serine-threonine kinase protein kinase D2 (PRKD2) as a potential effector of GABP in HSCs. Prkd2 expression was markedly reduced in Gabpα-null HSCs and progenitor cells. Reduced expression of PRKD2 or pharmacologic inhibition decreased cell cycling, and PRKD2 rescued growth of Gabpα-null BCR-ABL-expressing cells. Thus, GABP is required for HSC cell cycle entry and CML development through its control of PRKD2. This offers a potential therapeutic target in leukemia.

Effects of Rabbit Anti-Mouse Thymocyte Globulin Treatment in a Splenocyte-Induced Model of Aplastic Anemia

AbstractAbstract 1172Acquired aplastic anemia is an immune-mediated disease where destruction of hematopoietic stem cells (HSCs) in the bone marrow results in severe and life-threatening pancytopenia. Thymoglobulin® is often used as immunosuppressive therapy in this disease with up to 80 percent of patients responding to a combination of Thymoglobulin and cyclosporine. In an effort to better understand the activities and mechanism of action of Thymoglobulin we developed a mouse model of immune-mediated aplastic anemia and evaluated a murine surrogate of Thymoglobulin®, rabbit anti-mouse thymocyte globulin (mATG) in this model. We modified a graft-versus-host (GVH)-induced model described in the literature (Bloom, et al., 2004) utilizing HSC-depleted spleen cells transferred from C57BL/6 into CByB6F1 mice instead of lymph node cell transfer. Our modified model shows a cell dose-dependent increase in pancytopenia and lethality. Mice receiving a high dose (100×106) of HSC-depleted splenocytes experienced severe pancytopenia and rapid death occurring around day 21 whereas mice receiving lower doses (70×106, 35×106 and 17×106) of cells showed progressively less pancytopenia and lethality as the dose of cells decreased. Histopathology also showed marked loss of hematopoietic progenitor cells in the bone marrow with little evidence of GVHD in other tissues. Prophylactic administration of mATG (25mg/kg, 2x, day 0 and 3) to mice given high doses of HSC-depleted splenocytes (100×106) resulted in a significant improvement in pancytopenia and survival (70%) in this model. Interestingly, therapeutic administration of mATG was more effective when given later relative to disease induction. Delivery of mATG (25mg/kg, 2x, three days apart) starting on day 3 showed some delay in disease progression (day 30 vs day 21) and mATG started on day 6 slightly increased survival (40%). However, mice receiving mATG starting on days 10 or 14 showed a much greater overall survival of 100% and 60%, respectively, with full rebound of hematopoietic cells in the blood to normal levels. The complete response observed with later mATG administration (day 10 or day 14) mimics the treatment and response of patients given Thymoglobulin®. In summary, we have established a novel model of HSC-depleted splenocyte induction of bone marrow failure in mice that is responsive to therapeutic ATG administration. Studies in this model will aid in further understanding the mechanism of ATG in aplastic anemia and may contribute to the development of potential new therapies. Disclosures:Ruzek:Genzyme: Employment. Phillips:Genzyme: Employment. Richards:Genzyme: Employment. Mamlouk:Genzyme: Employment. Williams:Genzyme: Employment. Garman:Genzyme: Employment.

Defective proliferative capacity and accelerated telomeric loss of hematopoietic progenitor cells in rheumatoid arthritis

In rheumatoid arthritis (RA), telomeres of lymphoid and myeloid cells are age-inappropriately shortened, suggesting excessive turnover of hematopoietic precursor cells (HPCs). The purpose of this study was to examine the functional competence (proliferative capacity, maintenance of telomeric reserve) of CD34+ HPCs in RA. Frequencies of peripheral blood CD34+,CD45+ HPCs from 63 rheumatoid factor-positive RA patients and 48 controls matched for age, sex, and ethnicity were measured by flow cytometry. Proliferative burst, cell cycle dynamics, and induction of lineage-restricted receptors were tested in purified CD34+ HPCs after stimulation with early hematopoietins. Telomere sequences were quantified by real-time polymerase chain reaction. HPC functions were correlated with the duration, activity, and severity of RA as well as its treatment. In healthy donors, CD34+ HPCs accounted for 0.05% of nucleated cells; their numbers were strictly age dependent and declined at a rate of 1.3% per year. In RA patients, CD34+ HPC frequencies were age-independently reduced to 0.03%. Upon growth factor stimulation, control HPCs passed through 5 replication cycles over 4 days. In contrast, RA-derived HPCs completed only 3 generations. Telomeres of RA CD34+ HPCs were age-inappropriately shortened by 1,600 bp. All HPC defects were independent of disease duration, disease activity, and smoking status, and were present to the same degree in untreated patients. In RA, circulating bone marrow-derived progenitor cells were diminished, and concentrations stagnated at levels typical of those in old control subjects. HPCs from RA patients displayed growth factor nonresponsiveness and sluggish cell cycle progression; marked telomere shortening indicated proliferative stress-induced senescence. Defective HPC function independent of disease activity markers suggests bone marrow failure as a potential pathogenic factor in RA.

E2F-6 Suppresses Growth-Associated Apoptosis of Human Hematopoietic Progenitor Cells by Counteracting Proapoptotic Activity of E2F-1

E2F-6 is a dominant-negative transcriptional repressor against other members of the E2F family. In this study, we investigated the expression and function of E2F-6 in human hematopoietic progenitor cells to clarify its role in hematopoiesis. We found that among E2F subunits, E2F-1, E2F-2, E2F-4, and E2F-6 were expressed in CD34(+) human hematopoietic progenitor cells. The expression of E2F-6 increased along with proliferation and decreased during differentiation of hematopoietic progenitors, whereas the other three species were upregulated in CD34(-) bone marrow mononuclear cells. Overexpression of E2F-6 did not affect the growth of immature hematopoietic cell line K562 but suppressed E2F-1-induced apoptosis, whereas it failed to inhibit apoptosis induced by differentiation inducers and anticancer drugs. Among E2F-1-dependent apoptosis-related molecules, E2F-6 specifically inhibited upregulation of Apaf-1 by competing with E2F-1 for promoter binding. E2F-6 similarly suppressed apoptosis and Apaf-1 upregulation in primary hematopoietic progenitor cells during cytokine-induced proliferation but had no effect when they were differentiated. As a result, E2F-6 enhanced the clonogenic growth of colony-forming unit-granulocyte, erythroid, macrophage, and megakaryocyte. These results suggest that E2F-6 provides a failsafe mechanism against loss of hematopoietic progenitor cells during proliferation. Disclosure of potential conflicts of interest is found at the end of this article.

Optimization of purging of autologous bone marrow grafts for children with precursor B acute lymphoblastic leukemia.

In our laboratory, a two-step procedure is used for purging precursor B ALL from autologous bone marrow grafts of children in second bone marrow remission. An immunorosette depletion method with CD19 and CD22 MAbs is followed by one cycle of complement-mediated cell lysis with CD9 and CD10 MAbs. The aim of the present study was to determine if the efficacy of this procedure could be further enhanced by including CD20 and CD72 MAbs in the current protocol. Leukemia-contaminated remission bone marrow was simulated by mixing cell line cells and normal bone marrow cells. The efficacy of purging of malignant cells was determined by culturing the cells in a limiting dilution assay. The effect of including CD20 and CD72 in the immunorosette depletion was limited. In contrast, when these MAbs were added during complement-mediated cell lysis, a significant increase in depletion of tumor cells was observed. This was true when complement lysis was carried out alone (0.4 versus 3.0 log depletion for Ros cells) and when it was preceded by immunorosette depletion (2.7 versus 4.1 log depletion for Ros cells). The loss of hematopoietic progenitor cells was not greater than with the current purging protocol.

A combination of CD34 selection and complement-mediated immunopurging (anti-CD15 monoclonal antibody) eliminates tumor cells while sparing normal progenitor cells.

Autologous bone marrow transplantation (ABMT) for acute myeloid leukemia (AML) in first complete remission (CR) results in a prolonged disease-free survival (DFS) of 34%-57%. Relapse of the underlying disease is the major cause for failure of ABMT. Relapse can result fom tumor cells either surviving in the patient or reinfused in the autograft. Genetic marking of autografted cells has demonstrated that transplanted cells contribute to relapse. This finding supports the use of purged autografts. Several purging techniques have been used. Immunologic purging using the monoclonal antibody (mAb) PM-81 (anti-CD15) has been used by our center with a long-term DFS in 50% of AML patients. PM-81 reacts with 90% of AML patients, and we have used it for over 10 years. We have investigated a two-stage purging technique involving initial selection for CD34+ cells followed by mAb purging in bone marrow (BM) and peripheral blood stem cell (PBSC) harvests. This method achieved up to a 7 log diminution in leukemic cells and 1-4 log reduction in CD15+ cells, without a significant loss of hematopoietic progenitor cells. This double-purging technique has the advantages of cytoreduction, elimination of CD34- leukemic cells, and possible improvement in the clinical efficacy of purging by concentrating for CD34+ cells. Cytoreduction by CD34 enrichment followed by purging may facilitate the use of PBSC transplants in AML.

Separation, enrichment, and characterization of human hematopoietic progenitor cells from umbilical cord blood.

Human umbilical cord blood (UCB) may be used as an alternative source of bone marrow repopulating cells in allogeneic bone marrow transplantation in children. It has been reported that high numbers of hematopoietic progenitor cells (HPC) from umbilical cord blood may be lost during simple cell-separation techniques. This may seriously hamper the use of UCB as an alternative source of bone marrow repopulating cells. In this study we demonstrate that UCB can be separated into various cell fractions using several cell-separation methods including red blood cell lysis, methylcellulose sedimentation, and density gradients without significant loss of HPC, when cell separations are initiated within 8 h. We demonstrate that UCB contains a high concentration of immature HPC as compared with bone marrow grafts. Using FACS analysis of cells harvested from single colonies derived from single cell- single well-sorted CD34++ CD33- UCB cells, the high frequency of multipotential HPC was illustrated. These results suggest that UCB may contain sufficient HPC for hematopoietic stem cell transplantation in adults.

Separation of mononuclear bone marrow cells using the Cobe 2997 blood cell separator.

In the present study, we report the results of our evaluation of the use of the continuous-flow cell separator Cobe 2997 to isolate from human bone marrow (BM) aspirates the mononuclear cell (MNC) fraction containing hematopoietic stem cells. This MNC concentrate is isolated in 15% of the original BM volume and contains 23% of the initial nucleated cells. It is enriched as concerns the BM MNC fraction (lymphocytes + monocytes recovery; 80%), whereas the contamination with granulocytes, red blood cells and platelets is reduced to 7.2, 1.5% and 41%, respectively, of the cells initially present in the BM suspensions. Furthermore, it is demonstrated that this MNC concentrate is highly enriched in granulocyte-macrophage-colony-forming cells (CFU-GM; recovery 83%). The method is simple, inexpensive, efficient and reproducible. It allows rapid processing of a large volume of BM without substantial loss of hematopoietic progenitor cells. It represents a valuable method of BM MNC concentration prior to further in vitro manipulations such as T cell or tumor cell depletion or cryopreservation.

Separation of Mononuclear Bone Marrow Cells using the Cobe 2997 Blood Cell Separator

In the present study, we report the results of our evaluation of the use of the continuous-flow cell separator Cobe 2997 to isolate from human bone marrow (BM) aspirates the mononuclear cell (MNC) fraction containing hematopoietic stem cells. This MNC concentrate is isolated in 15% of the original BM volume and contains 23% of the initial nucleated cells. It is enriched as concerns the BM MNC fraction (lymphocytes + monocytes recovery; 80%), whereas the contamination with granulocytes, red blood cells and platelets is reduced to 7.2, 1.5% and 41%, respectively, of the cells initially present in the BM suspensions. Furthermore, it is demonstrated that this MNC concentrate is highly enriched in granulocyte-macrophage-colony-forming cells (CFU-GM; recovery 83%). The method is simple, inexpensive, efficient and reproducible. It allows rapid processing of a large volume of BM without substantial loss of hematopoietic progenitor cells. It represents a valuable method of BM MNC concentration prior to further in vitro manipulations such as T cell or tumor cell depletion or cryopreservation.

Enrichment of Marrow Hemopoietic Progenitor Cells using a Blood Cell Processor

A total of 93 bone marrows (BM) from normal donors and patients were processed using the IBM-COBE 2991 blood cell washer to produce a concentrated buffy coat (BC) for either bone marrow transplantation (BMT) or cryopreservation for subsequent autologous BMT. The reduction in volume was 73.3 +/- 8.5% and nucleated blood cells (NBC) recovery was 87.1 +/- 9.1% of original marrow. Red blood cell (RBC) and platelet (PLT) contamination was reduced 64.5 +/- 10.9% and 41.2 +/- 24.1%, respectively. Clonogenic activity indicated that the NBC fraction was highly enriched in hematopoietic progenitor cells (greater than 100%) as assessed in vitro (CFU-GM). Results were not affected by diagnosis, initial marrow volume or cell count of the BM suspension. We conclude that this is a simple and reproducible method using blood bank, facilities and permits BC preparation from BM without significant loss of hematopoietic progenitor cells.