Engraftment Of Hematopoietic Progenitor Cells Research Articles

In vitro vascular differentiation system efficiently produces natural killer cells for cancer immunotherapies

ABSTRACT Background Immunotherapeutic innovation is crucial for limited operability tumors. CAR T-cell therapy displayed reduced efficiency against glioblastoma (GBM), likely due to mutations underlying disease progression. Natural Killer cells (NKs) detect cancer cells despite said mutations – demonstrating increased tumor elimination potential. We developed an NK differentiation system using human pluripotent stem cells (hPSCs). Via this system, genetic modifications targeting cancer treatment challenges can be introduced during pluripotency – enabling unlimited production of modified “off-the-shelf” hPSC-NKs. Methods hPSCs were differentiated into hematopoietic progenitor cells (HPCs) and NKs using our novel organoid system. These cells were characterized using flow cytometric and bioinformatic analyses. HPC engraftment potential was assessed using NSG mice. NK cytotoxicity was validated using in vitro and in vitro K562 assays and further corroborated on lymphoma, diffuse intrinsic pontine glioma (DIPG), and GBM cell lines in vitro. Results HPCs demonstrated engraftment in peripheral blood samples, and hPSC-NKs showcased morphology and functionality akin to same donor peripheral blood NKs (PB-NKs). The hPSC-NKs also displayed potential advantages regarding checkpoint inhibitor and metabolic gene expression, and demonstrated in vitro and in vivo cytotoxicity against various cancers. Conclusions Our organoid system, designed to replicate in vivo cellular organization (including signaling gradients and shear stress conditions), offers a suitable environment for HPC and NK generation. The engraftable nature of HPCs and potent NK cytotoxicity against leukemia, lymphoma, DIPG, and GBM highlight the potential of this innovative system to serve as a valuable tool that will benefit cancer treatment and research – improving patient survival and quality of life.

Differential CXCR4 expression on hematopoietic progenitor cells versus stem cells directs homing and engraftment.

Gene therapy involves a substantial loss of hematopoietic stem and progenitor cells (HSPC) during processing and homing. Intra-BM (i.b.m.) transplantation can reduce homing losses, but prior studies have not yielded promising results. We studied the mechanisms involved in homing and engraftment of i.b.m. transplanted and i.v. transplanted genetically modified (GM) human HSPC. We found that i.b.m. HSPC transplantation improved engraftment of hematopoietic progenitor cells (HPC) but not of long-term repopulating hematopoietic stem cells (HSC). Mechanistically, HPC expressed higher functional levels of CXCR4 than HSC, conferring them a retention and homing advantage when transplanted i.b.m. Removing HPC and transplanting an HSC-enriched population i.b.m. significantly increased long-term engraftment over i.v. transplantation. Transient upregulation of CXCR4 on GM HSC-enriched cells, using a noncytotoxic portion of viral protein R (VPR) fused to CXCR4 delivered as a protein in lentiviral particles, resulted in higher homing and long-term engraftment of GM HSC transplanted either i.v. or i.b.m. compared with standard i.v. transplants. Overall, we show a mechanism for why i.b.m. transplants do not significantly improve long-term engraftment over i.v. transplants. I.b.m. transplantation becomes relevant when an HSC-enriched population is delivered. Alternatively, CXCR4 expression on HSC, when transiently increased using a protein delivery method, improves homing and engraftment specifically of GM HSC.

Enhanced Engraftment of Hematopoietic Progenitor Cells in Mice Treated With Granulocyte Colony-Stimulating Factor Before Low-Dose Irradiation: Implications for Gene Therapy

Gene therapy for inherited disorders of blood cells will require both efficient methods for stable gene transfer and nonablative bone marrow conditioning regimens to allow engraftment of modified hematopoietic progenitor cells (HPCs). We have used a sensitive murine system for detecting HPC engraftment using congenic C57BL/6 mice that differ at the Ly5 locus, which encodes the leukocyte common antigen. The system relies on the ability of monoclonal antibodies with specificity for Ly5.1 and Ly5.2 (revised nomenclature: CD45.1 and CD45.2, respectively) to distinguish donor and recipient peripheral blood leukocytes after transplantation of purified Sca-1+ bone marrow-derived HPCs. No detectable engraftment occurred in nonirradiated recipient mice, even when as many as 2.0 x 10(6) SCa-1+HPCs were transplanted. However, in mice receiving total body irradiation (TBI), engraftment increased as a function of pretransplantation radiation dose, number of transplanted cells, and time after transplantation. Moreover, mice receiving either granulocyte colony-stimulating factor (G-CSF) or G-CSF+ stem cell factor before low-dose TBI (160 cGy) exhibited a marked increase in engraftment compared with mice receiving a vehicle control before low-dose TBI (18.9% and 20.6% v 5.6% at a 1 month, respectively; 29% and 35% v 15.1% at 4 months, respectively). Use of growth factor pretreatment even allowed TBI doses as low as 30, 70, or 120 cGy to achieve significant engraftment of donor progenitors (0.3%, 1.5%, and 6.8% at 1 month, respectively; 1.7%, 5.8%, and 13.9% at 4 months, respectively). All animals remained healthy during the observation periods. Thus, growth factor preconditioning of the recipient followed by low-dose TBI may provide an optimal balance between safety and efficacy in achieving required levels of engraftment for gene therapy of blood disorders.

Phase II Trial of In Vivo Purging with Daratumumab in Newly Diagnosed Multiple Myeloma

Phase II Trial of In Vivo Purging with Daratumumab in Newly Diagnosed Multiple Myeloma

Viability and engraftment of hematopoietic progenitor cells after long-term cryopreservation: effect of diagnosis and percentage dimethyl sulfoxide concentration

We carried out a retrospective analysis of viability by diagnosis and dimethyl sulfoxide (DMSO) concentration in patients who had undergone autologous transplants using hematopoietic progenitor cells (HPC) after long-term storage (up to 17.8 years). Viability was tested using flow cytometry for HPC that were harvested and preserved using a controlled rate freezer and 5% or 10% DMSO with human serum albumin, then stored in liquid nitrogen. Data from 262 samples were analyzed (249 myeloma patients and 13 other diagnoses): 100 consecutively thawed samples with a storage time of <1 year (all 10% DMSO), 50 consecutive samples stored for 1-4.9 years (10% DMSO), 50 samples stored for 5-9 years (5% DMSO) and all samples stored and used for transplant after >9 years (60 samples, 5% DMSO; two samples, 10% DMSO). No statistically significant difference in viability between the 5% DMSO and 10% DMSO groups was observed (P = 0.08), so the 1-4.9 years and 5-9 years were combined and the three groups (<1 year, 1-9 years and >9 years) were compared using an anova test. There was no difference in viability based on cryostorage period (P = 0.23) or between myeloma and other diagnoses (P = 0.45). No difference was seen in time to White blood cell (WBC) engraftment (P = 0.10) or to platelet engraftment between groups (P = 0.52). These data suggest that long-term storage in 5% DMSO and human serum albumin is safe.

Viability and Engraftment of Hematopoietic Progenitor Cells (HPC) After Long Term Cryopreservation: Effect of Diagnosis and % Dimethylsulfoxide (DMSO) Concentration.

Abstract 3161Poster Board III-98 IntroductionThere are few reports of HPC viability after more than 9 y of storage. It has been suggested that HPC from myeloma patients (pts) may survive storage less well than those from other diseases. The appropriate concentration of DMSO for long term storage is unclear. Our program has collected and stored HPC since November 1989, changing from 5% DMSO to 10% DMSO in August 2000. We present a retrospective analysis of viability by diagnosis and DMSO concentration in pts who underwent autologous transplant using HPC after long-term storage (up to 17.8 y). Materials and MethodsHPC were harvested and preserved using a controlled rate freezer and 5% or 10% DMSO, then stored in liquid nitrogen. Viability was tested by flow cytometry using unwashed samples and looking at propidium iodide or 7-AAD uptake within the CD34+ population. Data from 242 samples were analyzed (224 myeloma pts and 18 other diagnoses): 100 consecutively thawed samples with storage time <1 y (all 10% DMSO), 100 consecutive samples stored from 5-9 y ( 7 samples, 10% and 93 samples, 5% DMSO), and all samples stored and used for transplant after >9y (42 samples, 5% DMSO). No statistically significant difference was seen in viability between <1y and 5-9y, so these groups were combined and compared to >9y. White blood cell (WBC) and platelet engraftment were compared for the 2 groups using an unpaired t test. ResultsMean viability for the 0-9y and >9 y groups were 91.62% and 92.79% respectively, with no statistically significant difference in viability between groups (p=0.28). Using a 2-tailed t test, no difference was seen in viability based on diagnosis (p=0.19) or between 5% and 10% DMSO (p= 0.12). No difference was seen in WBC engraftment (p=0.28) or platelet engraftment between groups (p=0.57). DiscussionHPC collections remained viable after 17.8 years, the longest time reported for use after storage to date. No difference in viability was seen with 5% or 10% DMSO, or between myeloma and other diseases. Storage time did not affect engraftment. DisclosuresNo relevant conflicts of interest to declare.

HOXB4-Transduced Embryonic Stem Cell-Derived Lin−c-kit+ and Lin−Sca-1+ Hematopoietic Progenitors Express H60 and Are Targeted by NK Cells

Embryonic stem (ES) cells are a novel source of cells, especially hematopoietic progenitor cells that can be used to treat degenerative diseases in humans. However, there is a need to determine how ES cell-derived progenitors are regulated by both the adaptive and innate immune systems post transplantation. In this study, we demonstrate that hematopoietic progenitor cells (HPCs) derived from mouse ES cells ectopically expressing HOXB4 fail to engraft long-term in the presence of NK cells. In particular, the H60-expressing Lin(-)c-kit(+) and Lin(-)Sca-1(+) subpopulations were preferentially deleted in Rag2(-/-), but not in Rag2(-/-)gamma(c)(-/-) mice. Up-regulation of class I expression on HPCs prevented their lysis by NK cells, and Ab-mediated depletion of NK cells restored long-term HPC engraftment. In contrast to the notion that ES-derived cells are immune-privileged, we show in this study that NK cells form a formidable barrier to the long-term engraftment of ES cell-derived hematopoietic progenitors.

Bone Marrow T Cells Are More Effective Than Peripheral T Cells in Inducing Chimeric Conversion Following MHC-Mismatched Nonmyeloablative Bone Marrow Transplantation

Background & Objectives: Recently, T cells in BM have attracted renewed interest because they are now known to have different surface phenotypes, subsets, and activation states from those in the periphery. Memory T cells undergo extensive migration from the blood to the BM and vice versa. The BM plays an important role in preferential homing and extensive proliferation of memory T cells, and contributes considerably to the longlived memory T cell pool. BM T cells are more activated than their splenic counterparts and have a higher rate of local proliferation. Although BM-T (NK1.1– CD4+ or CD8+) cells did not induce lethal GVH disease, even at high cell numbers, BM-T cells mediated vigorous graft-versus-tumor activity and facilitated engraftment of hematopoietic progenitor cells. These studies suggested that BM-T cells could be a useful cellular source for adoptive immunotherapy following ABMT, instead of peripheral T cells. Non-myeloablative bone marrow transplantation (NMT) and allogeneic mixed chimerism can provide an environment adequate for diminishing susceptibility to DLI-mediated GVHD and an immunological platform for DLI in both mouse and human models. In patients treated with DLI, a successful GVL effect is often associated with conversion to complete donor chimerism, supporting the concept of a graft-versus-host (GVH) response as part of the GVL effect. Thus, a quiet chimeric conversion following DLI is desirable to reach an optimal DLI-mediated GVL effect, without the occurrence of GVHD. Although in a mouse model, the administration of non-tolerant donor spleen cells to established mixed chimeras has been shown to convert mixed hematopoietic chimerism to full donor chimerism, without the concomitant development of GVHD, DLI in humans frequently results in serious GVHD and life-threatening complications. However, the use of BM-T cells, as compared with spleen T cells (SP-T), as the DLI source has not been investigated in allogeneic mixed chimerism prepared with NMT. In this study, we evaluated the beneficial alloreactivity of DLI using cryopreserved BM-T cells, a by-product obtained during the T cell depletion (TCD) procedure in BM grafting, to effectively induce chimeric conversion without the occurrence of GVHD in MHC-mismatched NMT.Methods: Cells were prepared using established procedures. During the T cell depletion (TCD) procedure in BM grafting, BM-T cells were obtained as a by-product and then cryopreserved for subsequent DLI using BM-T cells 21 days after the bone marrow transplant.Results: The administration of 5–10 × 105 BM-T (Thy1.2+) cells in mixed chimeras resulted in complete chimeric conversion, with self-limited graft-versus-host disease (GVHD) and no pathological changes. However, the administration of 5–10 × 105 SP-T (Thy1.2+) cells resulted in persistent mixed chimerism, with pathological GVHD signs in the liver and intestine.Conclusion: Our results suggest that DLI using BM-T cells, even in small numbers, could be more potent for inducing chimeric conversion in mixed chimerism than DLI using SP-T cells. Further study is needed to determine whether cryopreserved BM-T cells are an effective cell source for DLI to consolidate donor-dominant chimerism in clinical practice, without concerns about GVHD.

Ex vivo expanded hematopoietic progenitor cells improve cardiac function after myocardial infarction: Role of β-catenin transduction and cell dose

BackgroundCell-based therapy after myocardial infarction (MI) is a promising therapeutic option but the relevant cell subsets and dosage requirements are poorly defined. We hypothesized that cell therapy for myocardial infarction is improved by ex vivo expansion and high-dose transplantation of defined hematopoietic progenitor cells (HPCs). Methods and resultsSince β-catenin promotes self-renewal of stem cells we evaluated the therapeutic efficacy of β-catenin-mediated ex vivo expansion of mouse HPCs in a mouse model of myocardial ischemia/reperfusion followed by intraarterial cell delivery. The impact of cell dose was determined by comparing a low-dose (LD, 5×105 cells) vs. a high-dose (HD, 1×107 cells) cell transplantation regimen of β-catenin-HPCs. The impact of β-catenin modification of HPCs was determined by comparing control-transduced HPCs (GFP-HPCs) vs. transgenic β-catenin-HPCs. HD β-catenin-HPCs significantly improved LV function and end-systolic and end-diastolic dimensions as compared to saline and LD β-catenin-HPCs. Furthermore, while treatment with HD GFP-HPC resulted in a modest cardiac improvement the application of β-catenin-HPCs was superior, resulting in a significant improvement in EF, FS and LVESD over saline and control GFP-HPC treatment. Although myocardial engraftment of HPCs was only transient, as determined by cell quantification after dye labeling, β-catenin-HPC treatment significantly decreased infarct size, reduced cardiomyocyte apoptosis and increased capillary angiogenesis in vitro and in vivo. ConclusionEx vivo expanded HPCs improve cardiac function and remodeling post MI in a cell number- and β-catenin-dependent manner.

RhoH-Deficient Mice Show Altered B Cell Populations In Vivo.

RhoH is a GTPase-deficient, hematopoietic-specific member of the family of Rho GTPases (Li et al, 2002). RhoH has been described as regulating proliferation and engraftment of hematopoietic progenitor cells (Gu et al, 2005) and integrin-mediated adhesion in T cells (Cherry et al, 2004). Additionally, RhoH plays a critical role in T-cell development and T-cell receptor signaling (Gu et al, 2006; Dorn et al, 2007). However, the potential role of RhoH in the differentiation and biological functions of B cells are unknown. To answer these questions, we analyzed the B-cell phenotype of RhoH−/− mice and the in vitro properties of RhoH-deficient splenic B cells compared to their wild-type counterparts. RhoH−/− mice showed increased B-cell numbers in the bone marrow, mainly due to an increase in the number of pro-B, pre-B and immature B cells. In the spleen, lymph nodes and peripheral blood, RhoH−/− mice showed a significant decrease in the number of follicular (B-2) cells (B220+ CD93– IgDhigh CD21low). The number of splenic marginal zone B cells (B220+ CD93– IgDlow CD21high), plasma cells (CD93– CD38+ CD138+) in bone marrow and spleen, and B-1 cells (IgM+ CD5+) in peritoneal cavity were not significantly different from those in wild-type animals. These alterations have functional significance, since the serum concentrations of IgM and IgG1 were significantly lower in RhoH−/− mice. However, splenic B cells isolated from RhoH−/− mice did not show any significant differences in their in vitro activation by anti-IgM, CD40 ligation or IL-4 stimulation, nor did they differ in their proliferative response to lipopolysaccharide. In vitro migration of RhoH-deficient B cells in response to CXCL12 or CXCL13 was similar to that of wild-type B cells. Given the important role of RhoH in signal transduction downstream the T cell receptor, we investigated the possible role of RhoH in B cell receptor signaling. Although total splenic B cells from RhoH−/− mice showed markedly increased phosphorylation of SYK and ERK after anti-IgM stimulation compared to wild-type B cells, sorted populations of splenic B-2 and marginal zone B cells from RhoH−/− and wild-type animals did not differ in the activation of these kinases, suggesting that the observed difference can be attributed to the different cellular composition of the B cell compartment (i.e. B-2 vs marginal zone B cells) in RhoH−/− mice. These data imply that the phenotype observed in RhoH−/− mice may not reflect an intrinsic defect in B cells but may be attributed to crosstalk between B cells and other hematopoietic cell populations.Composition of B cell subsets in wild-type and RhoH−/− mice (total cell number ×106, ± standard deviation, N=9)Bone marrowSpleenRhoH+/+RhoH−/−RhoH+/+RhoH−/−total B cells7.8±1.811.0±2.4 (**)total B cells31.7±10.125.4±8.8pro-B0.12±0.030.15±0.04 (*)transitional8.7±1.28.6±2.8pre-B2.6±0.63.8±0.8 (***)B-211.6±4.17.6±2.5 (*)immature1.5±0.42.1±0.5 (*)marginal3.2±1.13.9±1.6mature1.4±0.71.7±0.9(*) indicates p<0.05; (**), p<0.01; (***), p<0.005

Cross-Talk between Rho GTPases Regulates Actin Cytoskeleton and Chemotaxis of Hematopoietic Progenitor Cell.

Movement of hematopoietic stem/progenitor cells into (engraftment) and out of (mobilization) the bone marrow involves actin cytoskeleton and chemotaxis. Members of the Rho GTPase family have been well known for their critical roles in morphogenesis and cell migration via regulating actin assembly. Loss of Rac1 and Rac2 alleles leads to defective engraftment and massive mobilization of hematopoietic progenitor cells (HPCs), which are associated with impaired chemotaxis and cortical filamentous (F)-actin polymerization (Gu et al., Science 302: 445–449). RhoH, a hematopoietic-specific member of the RhoE subfamily, negatively regulates HPC engraftment, chemotaxis, F-actin polymerization and Rac activities (Gu et al., Blood 105: 1467–1475). These findings suggest that RhoH may antagonize Rac function in regulating these cellular processes. However, molecular mechanism of the cross-talk between these Rho GTPases is not defined. In this study, we examined the role of RhoH in actin cytoskeleton organization, chemotaxis and Rac membrane translocation in response to stromal-derived factor 1α (SDF-1α) using RhoH-deficient HPCs and retrovirus-mediated expression of EGFP-fusion proteins.RhoH−/− HPCs exhibit increased migration in response to SDF-1α, especially at low concentration, as compared with wild-type (WT) cells [10ng/ml SDF-1α: 3.5 +/− 0.9 vs. 12.3 +/− 1.8; 100ng/ml SDF-1α: 21.4 +/− 1.7 vs. 32.3 +/− 3.4, migrated cells (%), WT vs. RhoH−/−, n=3, p< 0.01]. Migration without SDF-1α stimulation of RhoH−/− cells is also enhanced. RhoH−/− HPCs assemble cortical F-actin without SDF-1α stimulation, under conditions in which WT cells do not show F-actin polymerization [cells with F-actin (%): 8.9 +/− 0.9 vs. 72.8 +/− 4, WT vs. RhoH−/−, n=6, p<0.001]. Additionally, RhoH−/− HPCs exhibit increased active, GTP-bound Rac GTPases. PAK, a known downstream effector of Rac in regulating actin cytoskeleton, also shows hyperphosphorylation in RhoH-/− HPCs, suggesting that RhoH may regulate actin assembly and cell migration through Rac-mediated pathway. In support of this, expression of a dominant negative Rac1N17 mutant blocks cortical F-actin assembly in RhoH−/− cells [cells with F-actin (%): 60 +/− 1 vs. 19 +/− 7, EGFP-Rac1 vs. Rac1N17, n=2].To further address the mechanism by which RhoH cross-talks to affect Rac signaling, we examine the role of RhoH in subcellular localization of EGFP-Rac proteins. SDF-1α induces activation of Rac, leading to translocation to the cell membrane where it co-localizes with lipid rafts and mediates cortical F-actin assembly in HPCs. In contrast, the dominant negative Rac1N17 does not localize to the cell membrane after SDF-1α stimulation. In RhoH−/− HPCs, EGFP-Rac protein presents at the cell membrane in the absence of SDF-1α [cells with membrane-localized EGFP-Rac1 (%): 7.5 +/− 3.9 vs. 44.5 +/− 6.4, WT vs. RhoH−/−, n=2]. In contrast, overexpression of RhoH in HPCs blocks translocation to the cell membrane after SDF-1α stimulation of Rac1, Rac2 and active Rac1V12. Finally, we found that RhoH, a constitutively active, GTP-bound protein, preferentially localizes to the cell membrane even in the absence of SDF-1α. This localization is dependent upon the prenylation site and the c-terminal domains of RhoH. Lack of membrane localization is associated with defective biological function. Together, our data suggest that RhoH is essential for proper cortical F-actin assembly and chemotaxis of HPCs via regulating Rac activation and membrane localization, and implicates a functional cross-talk between RhoH and Rac.

Superior Bone Marrow Homing of G-CSF Mobilized Hematopoietic Progenitor Cells (HPC) - A Home Run.

The kinetics of HPC engraftment correlate to a significant degree with the efficiency of bone marrow (BM) homing. We recently documented that fresh BM-HPC, rendered SDF-1 unresponsive or Gi-signaling refractory, homed normally, due to efficient retention through α4-integrin/VCAM 1. However, when deletion of α4-integrin or VCAM-1 was combined with SDF-1/CXCR4- or Gi-protein-signaling blockade, BM homing was drastically reduced, uncovering non-dominant contributions of SDF-1/CXCR4 and Gi-proteins. When, in contrast, cytokine (SCF) incubated HPC were transplanted, loss of CXCR4- or Gi-signaling alone significantly reduced BM homing, with little compensation by α4/VCAM-1 and endothelial selectins. These studies depicted a flexible hierarchy of cooperating homing pathways, in which the dominance of the individual pathway contribution is shifted in response to the cytokine milieu. Based on these data, we reasoned that different donor transplant sources might differ in the relative contribution of each homing pathway. This concept was tested with G-CSF mobilized (MPB) and fresh steady-state BM (ssBM) HPC. To assess the homing properties of these two cell sources, we controlled the number of transplanted colony-forming cells (CFU-C). We found that murine or human MPB-HPC homed significantly better than the respective ssBM-HPC: 1.16±0.04% per femur vs. 0.80±0.03% for murine cells, and 1.64±0.13% vs. 0.51±0.04% for human cells transplanted to NOD/SCIDβ2microglobulin −/− recipients were recovered 18h after transplantation (n>20, p<0.005). A similar homing advantage was observed 3h and 48h after transplantation. To explore underlying mechanisms, several previously described differences between ssBM and MPB were rigorously addressed and correlated with in vivo homing. Despite down-regulated CXCR4, chemokinesis and SDF-1-directed chemotaxis of MPB-HPC were increased compared to fresh ssBM-HPC. Because of their enhanced motility, blocking of Gi-protein- or CXCR4-signaling reduced MPB-HPC homing by more than 50% (1.19±0.08% vs. 0.45±0.03% for Pertussis toxin treated (@18h), 0.87±0.07% vs. 0.39±0.04% for AMD3100 treated (@ 3h); p<0.005), similarly to cytokine-incubated BM-, but in contrast to fresh ssBM-HPC. We observed virtual loss of expression of CD26, a feedback inhibitor of SDF-1/CXCR4 signaling, from MPB c-kit+ cells, and inhibition of CD26 activity increased BM homing only of ssBM-HPC (from 0.85±0.04% to 1.05±0.05%, p<0.05, as described), but not of MPB-HPC (1.17±0.09% vs. 1.13±0.05%). These novel findings suggest that CD26 down-regulation on MPB cells and their exposure to a “circulatory environment” may contribute to improved migration and homing. Taken together, the data indicate: a) MPB display better homing compared to ssBM, correlating with their modified migratory behavior. b) SDF/CXCR4- and other Gi-signals make dominant contributions to MPB-HPC homing, in contrast to fresh ssBM. c) The recently proposed augmentation of BM homing/engraftment with CD26 inhibitors is ineffective for MPB, due to virtual absence of CD26 expression from MPB-HPC.

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.

RhoH, a hematopoietic-specific Rho GTPase, regulates proliferation, survival, migration, and engraftment of hematopoietic progenitor cells

AbstractRho guanosine triphosphatases (GT-Pases) are recognized as critical mediators of signaling pathways regulating actin assembly, migration, proliferation, and survival in hematopoietic cells. Here, we have studied a recently identified hematopoietic-specific Rho GTPase, RhoH. Unlike most members of the Rho GTPase family, RhoH is GTPase deficient and does not cycle between GTP- and guanosine diphosphate (GDP)–bound forms, suggesting that regulation of RhoH expression may be critical in its activity. We found that RhoH is expressed in murine hematopoietic progenitor cells (HPCs) and fully differentiated myeloid and lymphoid lineages. In cytokine-stimulated HPCs, knockdown of RhoH expression via RNA interference stimulates proliferation, survival, and stromal cell-derived factor-1α (SDF-1α)–induced migration in vitro. Conversely, RhoH overexpression in these cells via retrovirus-mediated gene transfer is associated with impaired activation of Rac GTPases, reduced proliferation, increased apoptosis, and defective actin polymerization and chemotaxis. In vivo, HPCs with RhoH overexpression demonstrate defective hematopoietic reconstitution capability compared with control vector-transduced cells. Our results suggest that RhoH serves as a negative regulator of both growth and actin-based function of HPCs possibly via suppression of Rac-mediated signaling.

RhoH, a Hematopoietic-Specific Rho Gtpase, Regulates Proliferation, Survival, Migration and Engraftment of Hematopoietic Progenitor Cells.

Rho GTPases are recognized as critical mediators of signaling pathways regulating actin assembly, migration, proliferation and survival in hematopoietic cells. Here, we have studied a newly identified hematopoietic-specific Rho GTPase, RhoH. The RhoH gene was first identified as a hypermutable locus in non-Hodgkin's lymphoma and diffuse large B-cell lymphoma, and alteration of RhoH expression has been implicated in some cases. Unlike most members of the Rho GTPase family, RhoH is GTPase deficient and remains in the active, GTP-bound state suggesting that the level of RhoH expression may be a critical determinant of activity. To elucidate the biological role of RhoH in blood cell development and function, we used two complementary approaches, including overexpression via retrovirus-mediated gene transfer and knock-down via RNA interference, to alter levels of RhoH in hematopoietic progenitor cells (HPCs) and examined the effects of altered RhoH expression on hematopoiesis. In cytokine-stimulated HPCs, knock-down of RhoH expression (to ~ 30% of the endogenous level) significantly induces proliferation, survival and stromal cell derived factor-1 α (SDF-1α)-induced migration and cortical filamentous (F)-actin assembly (Table I). Conversely, RhoH overexpression (>2 folds of the endogenous level) in these cells is associated with reduced proliferation and increased apoptosis in response to stem cell factor, and defective F-actin polymerization at cell peripheral membrane and chemotaxis in response to SDF-1 α (Table I). In the irradiated recipient mice, hematopoietic stem/progenitor cells overexpressing RhoH demonstrate defective reconstitution capability in peripheral blood (Table I), bone marrow, spleen and lymph node as compared with control vector-transduced cells. Also, the RhoH-transduced donor cells displayed an increased level of apoptosis and a reduced proliferative response to the 5-fluorouracil treatment when compared with vector-transduced cells. Interestingly, these defective phenotypes associated with the progenitor cells overexpressing RhoH resemble the phenotypes of Rac1-/- ; Rac2-/- cells. Overexpression of RhoH is also associated with reduced Rac activity when stimulated by a Rac agonist. In addition, overexpression of RhoH disrupts the SDF-α-stimulated subcellular redistribution of Rac1 and Rac2 1 to cell peripheral membrane where Rac proteins are activated and induce F-actin polymerization. These results suggest that aberrant expression of RhoH leads to abnormal growth and actin-based function of HPCs. RhoH functions as a negative regulator of proliferation, survival, migration, actin polymerization and engraftment of HPCs possibly through suppression of Rac activation via preventing localization of Rac proteins to cell peripheral membrane.Table 1. Cellular phenotypes associated with the altered expression levels of RhoH in HPCs.Proliferation (CPM x 1000)Apoptosis (% Annexin V+ cells)Migration (% migrated cells)F-actin (% cells with cortical F-actin staining)engraftment (%EGFP+ cells in PB)vector control16.5 +/− 2.03.2 +/− 0.822.0 +/− 2.07538.0 +/− 6.0HA-RhoH7.0 +/− 0.8 *6.5 +/− 1.1*10.5 +/− 1.3 *2218.0 +/− 3.5 *siRhoH23.0 +/− 4.5 **1.8 +/− 0.4 **32.0 +/− 4.8 *95NA* p < 0.01; ** p < 0.05, HA-RhoH- and siRhoH-transduced vs vector-transduced cells.

Promotion of xenogeneic hematopoietic chimerism in rodents by mononuclear phagocyte depletion.

The successful establishment of tolerance toward pig tissues in primates through hematopoietic progenitor cell engraftment is restricted by the rapid disappearance of these cells in the recipient following infusion. We developed and tested the hypothesis that phagocytes of the reticuloendothelial system are responsible for the rapid clearance of infused pig hematopoietic cells using a mouse model. Mice received non-myeloablative conditioning and, on various days, were injected with medronate-encapsulated liposomes (M-L) or control blank liposomes, followed by the intravenous infusion of miniature swine hematopoietic cells. M-L were well-tolerated in mice (n=100) at levels that deplete mononuclear phagocytes. Depletion of mononuclear phagocytes in normal Balb/c mice as well as in severe combined immune deficient mice increased the accumulation of pig hematopoietic cells in the bone marrow (BM) by 10-fold when measured 24 h after the infusion of the cells. Colony-forming unit analysis showed an increased accumulation of pig hematopoietic progenitors in the BM of mice that were infused with medronate-liposomes. We conclude that depletion of mononuclear phagocytes by M-L has the potential to lower the barrier to the establishment of mixed chimerism and tolerance induction in xenotransplantation.