Hematopoietic Stem And Progenitor Cells Transplantation Research Articles

Deletion of Fak in Hematopoietic Stem Cells Leads to Enhanced Engraftment.

Hematopoietic stem and progenitor cells (HSPC) can be induced to leave the bone marrow (BM) and such mobilized hematopoietic stem cell (HSC) enriched cell populations when infused intravenously rapidly home back to the marrow and transmigrate into the extravascular compartment where they lodge in specialized niches. Niche induced signals coupled to intrinsic factors, promote HSC maintenance and regulate HSC localization, migration and/or differentiation. A better understanding of molecular pathways governing these HSC fate decisions could identify new targets for ex vivo HSC expansion and improvement of transplantation. Focal adhesion kinase (Fak) is a non-receptor protein tyrosine kinase, whose function has been well studied in fibroblasts, where it plays an important role in cell adhesion, survival and motility. Fak is also differentially expressed in HSPC, with highest expression in long-term HSCs (LT-HSCs, Lin-Sca-1+c-Kit +CD34-). Its functional role in hematopoiesis has not been well studied due to early embryonic lethality (E.8) of Fak−/− mice. We have generated conditional Fak deleted mice (Fakfl/flMx1-Cre+) to define how Fak signaling contributes to different components of HSC transplantation. We observed that the total cellularity in BM was comparable between Fak knockout (Fak−/−Mx1-Cre+) and control (Fakfl/fl) mice. The frequencies of both Lin-Sca-1+c-Kit + cells (LSKs) and short-term HSCs (ST-HSCs, Lin-Sca-1+c-Kit +CD34+) increased 2-fold in BM of Fak knockout mice compared to WT controls (n=5; p<0.05), while LT-HSCs appeared unaffected. Moreover, the number of LSKs in the spleens of Fak knockout mice increased by 3-fold relative to controls (n≥7, P<0.01). In vitro colony-forming assay also showed that the functionally defined progenitor cell numbers significantly increased in Fak knockout mice spleen. However, Fak deletion did not affect HPC populations including common myeloid progenitors (CMP), granulocyte macrophage progenitors (GMP), megakaryocyte erythrocyte progenitors (MEP) and common lymphoid progenitors (CLP). Importantly, in the competitive repopulation assay Fak−/− HSCs exhibited 2-fold enhanced engraftment for 20 weeks post-transplant, while lineage reconstitution in peripheral blood (PB) was similar between wild-type and knockout donor-derived cells suggesting Fak deletion did not affect PB mature B, T and myeloid cell survival under non-stress conditions. These findings indicate that FAK may play an important role in HSC homeostasis and that targeting the FAK pathway ex vivo may lead to innovative approaches in human HSPC transplantation.

Spatial Analysis of Hematopoietic Stem and Progenitor Cells in the Bone Marrow

Sustained production of all mature blood cell types relies on the continuous proliferation and differentiation of a rare population of self-renewing, multipotent hematopoietic stem cells (HSCs). HSC maintenance and lineage differentiation are strictly regulated by distinct microenvironments, termed niches, defined by cellular components, soluble regulators, and by the extracellular matrix. Definitive identification of the location as well as cellular and extracellular characteristics of HSC niches in the bone marrow (BM) has not been completed due to limitations of conventional imaging techniques. We have employed a novel imaging technology, Laser Scanning Cytometry (LSC) to define the localization of hematopoietic stem and progenitor cells (HSPCs) within different regions of the BM. LSC allows imaging and objective quantitative analysis of the anatomical position(s), number, and frequency of specific cell populations within the native tissue microenvironment. Analysis of whole femoral longitudinal sections of Bmi-GFP mice, in which GFP is expressed at its highest levels in HSPCs, revealed that within the bone diaphysis, HSPCs (Bmi-GFPhi c-kit+) cells were highly enriched in endosteal regions (within 100nm away from inner bone surface) compared to the central medullary region. Importantly, our data show that HSPCs are found at highest frequencies in the metaphysis of long bones, suggesting that these areas, which display characteristic morphological features, are functionally distinct from the diaphyseal region and a preferential location for HSPC-specific niches. We are currently employing LSC to identify HSPC niche cellular constituents by quantifying the relative frequency at which these cells are found in association with previously proposed niche-components such as osteoblasts, BM endothelial sinusoidal cells and CXCL12-abundant reticular cells. A detailed understanding of niche-derived signals regulating unique properties of HSCs will certainly prove relevant in human HSPC transplantation and cell therapy.

Peripheral blood hematopoietic stem cells: Biology, apheresis collection and cryopreservation

ilematopoiesis is a continuous, dynamic and highly complex process resulting in production of various mature blood cells from a small population of pluripotent stem and progenitor cells through diverse proliferative and differentiative events. Numerous studies have demonstrated that a complex network of interactive cytokines regulates the survival, maturation, and proliferation of hematopoietic stem and progenitor cells. APPLICATION OF CELL-MEDIATED THERAPY: Massive application of different cell-mediated therapeutic methods has resulted in an increased need for both specific IISPCs and operating procedures providing minimal cell damage during collection, processing and storage in a liquid or fiozen state. Therefore, the basic aim of cell harvesting, selection, as well as cryopreservation is to minimize cell damage during these procedures. HSPCs are cells which exhibit extensive self-renewal and proliferative capacity, associated with the capacity to differentiate into all blood cells and other cell lineages (plasticity of HSPC). Thanks to these properties, stem cells can provide complete and permanent restoration of hematopoiesis, which is the basis for clinical employment of HSPC transplantation. In addition, totipotent stem cells can be used for the so called "cell-therapy" in different clinical settings (e.g. myocardial regeneration after acute infarction). Despite the fact that HSPC transplantation is already in routine use, some questions related to the optimal blood progenitor/cell collection, selection, storage and clinical use are still unresolved. Therefore, this review only briefly discusses the therapeutic use of HSPCs in different clinical areas and focuses on the recommendations, as well as the specific transfusion policies related to HSPC collection, processing, and cryopreservation with an emphasis on quality control.

Aldehyde Dehydrogenase (ALDH) Activity Resolves Neuroblastoma (NB) Cells and Hematopoietic Stem and Progenitor Cells (HPC) in Graft Material: Potential Method for Clinical Purging.

High dose, myeloablative chemotherapy followed by autologous HPC transplantation is used clinically to treat NB in children. The presence of NB cells in transplant material adversely affects outcome. Grafts have been purged by using monoclonal antibodies to select CD34+ HPC for transplantation or to remove tumor cells expressing NB surface antigens from graft material. We are exploring the feasibility of purging NB autografts by flow sorting HPC and NB cells based on differential expression of ALDH. Cell populations with high levels of ALDH activity and low side scatter (ALDHbrSSClo cells) can be identified in mobilized peripheral blood (PBSC), bone marrow (BM), and umbilical cord blood using the ALDEFLUOR™ [StemCo Biomedical, Inc.] method for flow cytometric measurement of cellular ALDH activity. ALDHbrSSClo cells isolated by flow sorting are highly enriched for HPC activity. We report here that none of three established NB cell lines (SK-N-SH, SK-N-BE(2), and IMR-32) express detectable ALDH activity in the ALDEFLUOR™ assay. Furthermore, when any of these NB lines was labeled with the stable, red fluorescent intracellular marker SNARF™ [Molecular Probes, Inc.] and then mixed with BM or PBSC, NB were readily resolved from the green fluorescent ALDHbrSSClo cells. In mixtures containing equal numbers of tumor cells and PBSC or BM cells 0–6 events were detected in the ALDHbrSSClo region out of a total of 3-10 x 105 events in the SSC vs. FSC gate. Even when mixtures containing 9 tumor cells to 1 leukocyte were used, fewer than 21 SNARF-labeled NB were detected in the ALDHbrSSClo region out of a total of 2-9 x 105events in the SSC vs. FSC gate. Additional studies were done by sorting mixtures of SNARF-labeled NB cells and PBSC or BM and measuring tumor burden post-sort. In a representative study, pre-sort NB content of 30% was reduced to 0.04% when care was taken to eliminate NB-HPC doublets by using pulse width discrimination; without this precaution, NB contamination in the sorted cells was 1.3%. RNA was prepared from pre- and post-sorted cells, and real time PCR analysis was performed using probes to NB gene products tyrosine hydroxylase, GD2 synthase and PRAME. PCR signals were readily detected in the starting sample and were reduced by >1000-fold in the sorted ALDHbrSSClo cells. To extend these findings with NB cell lines to clinical material, a bone marrow sample was obtained from a NB patient following appropriate institutional consent procedures and was analyzed by multiparameter flow analysis using ALDEFLUOR™ and a monoclonal antibody to GD2 synthase, an antigen strongly expressed by NB cells. Approximately 60% of the cells in this BM sample expressed GD2 synthase, but only 0.03% of the NB cells were detected among ALDHbrSSClo events. RNA was prepared from 100,000 flow-sorted ALDHbrSSClo cells, and real-time PCR analysis was performed. NB gene products were readily detected in the donor BMs, but no signal was detected in the sorted cell preparations. The NB burden in the flow-sorted ALDHbrSSClo population was reduced >100-fold compared to the unpurged, heavily contaminated BM. We are extending this analysis to maximize purging obtainable with clinical material. As ALDEFLUOR™ does not impair any tested functional characteristics of HPC, flow sorting based ALDH activity may provide a new method to purge autografts of NB cells for clinical transplantation.

SDF-1α-induced intracellular calcium transient involves Rho GTPase signalling and is required for migration of hematopoietic progenitor cells

Signalling through the chemokine stromal derived factor (SDF)-1α and its receptor CXCR4 has been recognized as a key event in the migratory response of hematopoietic stem and progenitor cells (HPC). Small GTPases of the Rho/Rac family might be involved in SDF-1α signalling at several different levels. In the present study we report that two toxins from Clostridium species which inhibit the small GTPase Rho suppressed SDF-1α-induced generation of intracellular calcium transients in HPC. Chelation of intracellular Ca 2+ with BAPTA or depletion of intracellular Ca 2+ stores with thapsigargin demonstrated that calcium transients are essential for SDF-1α-induced chemotactic migration of HPC. Furthermore, transplantation of HPC pretreated with Ca 2+ flux inhibitors into mice revealed a suppression of HPC homing to the bone marrow and increased levels of cells remaining in the bloodstream or circulating to the spleen. Our data indicate that the small GTPase Rho is required for the induction of Ca 2+ transients in HPC, which in turn are necessary for the coordinated migratory response of HPC both in vitro and in vivo.

Marrow engraftment of hematopoietic stem and progenitor cells is independent of Gα i-coupled chemokine receptors

The mechanism of hematopoietic stem and progenitor cell (HSPC) homing to hematopoietic organs after transplantation is still poorly understood. There is evidence that HSPC homing is a multistep process involving integrins and other adhesion molecules as well as stimulation of cytokine and chemokine receptors, similar to the process of lymphocyte recirculation and leukocyte emigration. This study examined the effect of pertussis toxin (PT), an inhibitor of signaling by many G α i protein-coupled chemokine receptors, on engraftment of HSPC. An in vitro incubation of total bone marrow cells in PT-supplemented media prior to transplantation into lethally irradiated syngeneic mice resulted in an increase in marrow repopulation and a parallel decrease of colony-forming unit-spleen (CFU-S) on day 13. PT treatment of Rh lowLineage negSca-1 pos cells prior to transplant resulted in delayed spleen cell engraftment, but no observable difference in the bone marrow cellularity compared to animals transplanted with untreated cells. FACS analysis of hematopoietic organs revealed that myeloid cell recovery in the bone marrow was unaffected by PT treatment of HSPC. However, a reduced myeloid cell recovery in the spleen and an increased B lymphoid recovery in both the spleen and the bone marrow were observed in recipients of PT-treated grafts relative to untreated grafts. To test the hypothesis that PT inhibits proliferation rather than engraftment of HSPC in the spleen, the effect of PT on cytokine-stimulated proliferation of HSPC was tested. Although an inhibition of the growth of microcolonies in response to interleukin 6 as a single cytokine could be observed after PT treatment, colony growth of HSPC after steel factor or steel factor + interleukin 6 stimulation was unaffected by PT. This study demonstrates that bone marrow, but not splenic, recovery after HSPC transplantation is independent of PT-sensitive mechanisms. It is likely that PT inhibits spleen cell recovery by disrupting a Gα i-coupled homing receptor expressed by HSPC. These studies support the hypothesis that distinct mechanisms regulate splenic vs bone marrow engraftment of HSPC, and that B lymphocyte progenitors and HSPC can utilize a PT-resistant homing mechanism to localize in hematopoietic tissues after transplantation.