Engraftment Of CD34 Research Articles

Targeting of human CD34(+) hematopoietic stem cells with myosin light chain preserves cardiac function in chronic infarcted mouse heart

Background We demonstrated that transplantation of the engineered CD 34(+) hematopoietic stem cells [CD34(+)HSC] mitigates cardiac function loss in damaged mouse hearts. Objective: To determine 1) if targeted arming CD34(+)HSC with bivalent antibodies directed against myosin light chain (MLC) antigen increases its homing in myocardial infarction (MI); 2) if engineered CD34(+)HSC mitigates functional loss in mouse MI. Methods: MI was created by ligation of the left descending artery in adult mice. After 48 hr, animals were divided into 3 groups: 1) MI (n = 4): animals received RPMI medium; 2) MI + CD34(+)HSC (n = 7): animals received 5x105 CD34(+)HSC; 3) Sham (n = 4): animals underwent thoractomy without MI. Two weeks later, cardiac contractility was measured in Langendorff model. Immunohistochemistry (IHS) was used to monitor the homing of armed CD34+HSC. Results: MI heart exhibited enlarged chamber diameters and a decrease in contractility comparedwith sham hearts. IHS demonstrated an accumulation of CD34+HSC in MI hearts. Engraftment of CD34+HSC increased left ventricular developed pressure (Sham/MI/CD34(+)HSC = 95 ± 1/58 ± 12/99 ± 5 mmHg, *p<0.05), rate pressure product (Sham/MI/CD34(+)HSC = 44 ± 3 x10−3/24 ± 5 x10−3/43 ± 5 x10−3 mmHg.beats/min, *p<0.05), heart rate (Sham/MI/CD34(+)HSC = 475 ± 36 /405 ± 32/432 ± 33 beats/min). Conclusion: Targeted arming of CD34+HSC with MLC results in an increase in contractility in post MI heart. The protective effects of CD34(+)HSC in MI is associated with an increase in accumulation of engineered CD34+HSC in MI hearts. (NIH 1 P20 RR018728)

Transplantation of Human Hematopoietic Stem Cells into Ischemic and Growing Kidneys Suggests a Role in Vasculogenesis but Not Tubulogenesis

Transplantation of murine bone marrow-derived stem cells has been reported recently to promote regeneration of the injured kidney. We investigated the potential of human adult CD34(+) progenitor cells to undergo renal differentiation once xenotransplanted into ischemic and developing kidneys. Immunostaining with human-specific antibodies for tubular cells (broad-spectrum cytokeratin), endothelial cells (CD31, PECAM), stromal cells (vimentin), and hematopoietic cells (pan-leukocyte CD45) demonstrated that although kidney ischemia enhanced engraftment of human cells, they were mostly hematopoietic cells (CD45(+)) residing in the interstitial spaces. Few other engrafted cells demonstrated an endothelial phenotype (human CD31(+)in morphologically appearing peritubular capillaries), but no evidence of tubular or stromal cells of human origin was found. Upregulation of SDF1 and HIF1 transcript levels in the ischemic kidneys might explain the diffuse engraftment of CD45(+)cells following injury. Similarly, when embryonic kidneys rudiments were co-transplanted with human CD34(+)cells in mice, we found both human CD45(+)and CD31(+)cells in the periphery of the developing renal grafts, whereas parenchymal elements failed to stain. In addition, human CD34(+)cells had no effect on kidney growth and differentiation. This first demonstration of human CD34(+)stem cell transplantation into injured and developing kidneys indicates that these cells do not readily acquire a tubular phenotype and are restricted mainly to hematopoietic and, to a lesser extent, to endothelial lineages. Efforts should be made to identify additional stem cell sources applicable for kidney growth and regeneration.

783. Transient Transduction of Repopulating Human CD34 Cells Using Nanoparticles in a Clinically Applicable Ex Vivo Protocol

Top of pageAbstract Transient transduction of human hematopoietic stem cells with novel nano-sized iron particles offers a new approach to transient cell modulation in vitro and in vivo. Moreover, nano particles serve a dual purpose by enabling tracking of labeled cells in vivo by means of magnetic resonance imaging. However, defining ex vivo cultivation strategies for the transduction and/or labeling of human HSC using these particles has not yet been well investigated. Here we present in vitro and in vivo data on human cord blood CD34+ HSC subjected to a 24 hr. clinically applicable ex vivo transduction protocol including protamine complexed ferrumoxide nanoparticles conjugated to Alexa 647 dye (FE-PRO[647]). Cell cultivation were carried out in tissue culture-treated 6 well plates coated with the CH-296 fibronectin fragment. We used X-Vivo 15 defined serum free media supplemented with 10 ng/ml rhTPO, rhSCF, and Flt-3-ligand. Initial CFU-GEMM data indicated that the presence of the FE-PRO [647] did not compromise the human HSC ability to generate colonies in vitro. Subsequent transplantation of labeled human cord blood CD34+ into beta2-NOD/SCID mice for in vivo tracking using flow cytometry consistently showed FE-PRO[647] labeled CD34+ cells in the marrow of the recipients three weeks post transplantation. The total human CD45 engraftment as evaluated in total murine marrow was 18.7+/|[minus]|11.3% (N=7), with a CD34+ component of 10.2+/|[minus]|9.0% (N=7). All 7 animals in the cohort were positive for FE-PRO[647] positive engrafted human cells (0.9+/|[minus]| 0.2 %, N=7). Most interestingly, a striking correlation of the total FE-PRO[647] positive cells and the CD34+ HSC was found in the marrow of the recipients (FE-PRO[647]+/CD34+ was 0.8+/|[minus]|0.2, N=7). Though a subset of CD133+ cells was found in all engrafted mice, interestingly enough no correlation of FE-PRO[647] and CD133+ was found. Moreover, correlating the mean fluorescence activity of the initially FE-PRO [647] transduced CB CD34+ cells to the FE-PRO[647]+/ CD34+ cells found in the recipient marrows 3 weeks post transplantation (relative value: 90.5 versus 2.9+/|[minus]|0.7, N=7) indicated that these CD34+ cells had only divided a total of 5 times over the 3 weeks time period. We hypothesize that these CD34+ cells represent long term engrafting human HSC, whereas the majority of the resulting CD34+ engraftment represents committed progenitors, giving rise to short term engraftment. Overall, these data show that nano particles can indeed be used to transiently transduce repopulating human HSC. This has lead us to define conditions for in vivo modulation of human HSC by co-transduction of FE-PRO[647] and the HIVEF1-EGFP lenti viral vector, with the aim of improving the lenti viral transduction efficiency. In vitro and in vivo data will be presented.

Co-Transplantation of Hematopoietic Stem Cells and GM-CSF/G-CSF/CSF-Transfected Mesenchymal Stem Cells in SCID Mice.

Mesenchymal stem cells (MSC) are multipotent and believed to facilitate the engraftment of hematopoietic stem cells (HSC) when transplanted simultaneously in animal studies and recently even in human trials. In this study, we transfected culture-expanded MSC with GM-CSF, G-CSF, and CSF cytokine genes and then co-transplanted with HSC to further promote HSC engraftment. Mononuclear cells were harvested from the various sources and seeded in long-term culture for ex vivo MSC expansion. The phenotype and purity of MSC were assessed by flow cytometry. We transferred the above three cytokine genes into ex vivo expanded MSC, confirmed transfection by fluorescent microscope of GFP, and thereafter did co-transplantation with HSC. A total of 1x107 HSC plus MSC/uL were introduced to tail vein of SCID mice. After 3–7 weeks later, with venous blood from the eyeballs, homing and engraftment of human cells were determined by flow cytometry and fluorescence in situ hybridization (FISH) studies. The total nucleated cell count and the engraftment of CD45+/CD34+ cells and XX/XY-positive human cells significantly increased in co-transplanted mice and even higher with the cytokine gene-transfected MSC in the order of GM-CSF, SCF, and G-CSF transfections (P<0.05). These results suggest that MSC transfected with hematopoietic growth factor genes are capable of enhancing the hematopoietic engraftment. Now we are planning to deliver genes involved in homing and cell adhesions, e.g., CXCR4, VLA, or TPO into ex vivo expanded MSC and do co-transplantation with HSC to further increase the efficiency of stem cell transplantation.

Extracellular UTP Affects In Vitro Migration and In Vivo Homing and Engraftment of CD34+ Hematopoietic Stem Cells.

Homing and engraftment of hematopoietic stem cells (HSCs) depend on the complex interplay between chemokines, cytokines, growth factors and adhesion molecules in the intricate architecture of bone marrow (BM) microenvironment. We have recently reported that CD34+ HSCs express P2Y and P2X receptors (P2Rs) for extracellular nucleotides. P2Rs activation by ATP and UTP produced stimulatory effects, in synergy with other hematopoietic cytokines, on clonogenic CD34+ and lineage-negative CD34− progenitor cells. ATP and UTP also expanded the more primitive CD34+- derived long-term culture-initiating cells. Extracellular nucleotides are also emerging as chemotactic factors for different cell types, including vascular endothelial cells, arterial smooth muscle cells and neutrophils, and regulate the trafficking of specific dendritic cell populations. In this study we asked if the extracellular nucleotide UTP modulates the migration of HSCs in response to the chemotactic peptide CXCL12/SDF-1α. Very low concentrations of UTP (10 μM) significantly improved stem cell migration, assayed with the dual-chamber migration system and evaluated by cell counting and clonogenic assays. Phenotypical analysis of CXCR4 antigen showed that SDF-1α gradients mainly attract HSCs expressing high levels of this receptor. Conversely, UTP induced the migration of HSCs expressing low levels of CXCR4. Furthermore, we evaluated actin polymerization as index of HSCs capacity to respond to chemotactic stimulation and to home to the BM microenvironment. The combined stimulation of CD34+ cells with UTP and SDF-1α produced a synergistic increase in actin polymerization, as indicated by the enhanced fluorescence intensity of FITC-phalloidin. Adhesion assays, performed in fibronectin-coated wells, also showed that UTP can stimulate the adhesion of HSCs to fibronectin, a key component of the extracelluar bone marrow microenvironment, without any substantial change in VLA-4 and VLA-5 expression levels. Moreover, xenogenic transplant studies showed that short-term pre-incubation with UTP significantly increased the engraftment efficiency of CD34+ HSC in nonobese diabetic / severe combined immunodeficiency mice. Taken together, our data suggest that UTP may affect HSC migration and homing to the BM, in synergy with the chemotactic peptide SDF-1α, perhaps activating a CXCR4-indipendent pathway.

Heterologous cells cooperate to augment stem cell migration, homing, and engraftment

AbstractT-lymphocyte depletion of bone marrow grafts compromises engraftment, suggesting a facilitating mechanism provided by the T cells that has been shown to associate with CD8+but not CD4+T cells. Explanations for this phenomenon have focused on immune targeting of residual host cells or cytokine production. We provide evidence for an alternative mechanism based on cooperative effects on cell motility. We observed that engraftment of CD34+cells in a β2-microglobulin–deficient nonobese diabetic/severe combined immunodeficiency (β2m−/−NOD/SCID) mouse model paralleled clinical observations in humans, with an enhancing effect noted from the addition of CD8+cells but not CD4+cells. This correlated with CD8+augmentation of CD34+cell homing to the bone marrow in vivo and CD8+cell–associated increases of CD34+cell transmigration through a bone marrow endothelial cell line in vitro. The cooperative interaction was not sensitive to brefeldin A inhibition of protein secretion. However, cytochalasin D–induced inhibition of CD8+cytoskeletal rearrangements abrogated CD34+transendothelial migration and impaired CD34+cell homing in vivo. CD8+cells did not migrate in tandem with CD34+cells or alter endothelial barrier integrity; rather, they affected phosphotyrosine-mediated signaling in CD34+cells in response to the chemokine stromal derived factor-1α (SDF-1α). These data demonstrate cell-cell cooperativity between different cell types in mediating chemotactic events and provide one potential explanation for the clinically observed effect of CD8+cells on bone marrow transplantation. This modification of cell migration by neighboring cells provides broad possibilities for combinatorial effects between cells of different types to influence cell localization.

Recent developments in the regulation of peripheral blood stem cell mobilization and engraftment by cytokines, chemokines, and adhesion molecules.

Peripheral blood stem cells (PBSC) have become the preferred source of stem cells for autologous transplantation because of the technical advantage and the shorter time to engraftment. Administration of hematopoietic growth factors such as granulocyte colony-stimulating factor (G-CSF) or granulocyte-macrophage colony-stimulating factor (GM-CSF) results in mobilization of PBSCs into the peripheral blood. G-CSF and GM-CSF differ somewhat in the number and composition of CD34(+) cells and effector cells mobilized to the peripheral blood; however, the molecular mechanism underlying the release and engraftment of CD34(+) cells by these growth factors is poorly understood. This review provides a recent update on the involvement of hematopoietic growth factors, chemokines, adhesion molecules, and chemokine receptors in the regulation of stem cell release and engraftment. The involvement of very late antigen-4 (VLA-4), VLA-5, leukocyte function associated-1 molecule (LFA-1), and L-selectin and their receptors CXCR4 and its ligand SDF-1 will be discussed, and cross talk between these factors will also be reviewed in the context of stem cell release and engraftment. Finally, PBSC mobilization by chemokines will be reviewed in relation to hematopoietic growth factors.

Efficient transduction of human hematopoietic repopulating cells generating stable engraftment of transgene-expressing cells in NOD/SCID mice

In an attempt to develop efficient procedures of human hematopoietic gene therapy, retrovirally transduced CD34+ cord blood cells were transplanted into NOD/SCID mice to evaluate the repopulating potential of transduced grafts. Samples were prestimulated on Retronectin-coated dishes and infected with gibbon ape leukemia virus (GALV)-pseudotyped FMEV vectors encoding the enhanced green fluorescent protein (EGFP). Periodic analyses of bone marrow (BM) from transplanted recipients revealed a sustained engraftment of human hematopoietic cells expressing the EGFP transgene. On average, 33.6% of human CD45+ cells expressed the transgene 90 to120 days after transplantation. Moreover, 11.9% of total NOD/SCID BM consisted of human CD45+ cells expressing the EGFP transgene at this time. The transplantation of purified EGFP+ cells increased the proportion of CD45+ cells positive for EGFP expression to 57.7% at 90 to 120 days after transplantation. At this time, 18.9% and 4.3% of NOD/SCID BM consisted of CD45+/EGFP+ and CD34+/EGFP+ cells, respectively. Interestingly, the transplantation of EGFP− cells purified at 24 hours after infection also generated a significant engraftment of CD45+/EGFP+ and CD34+/EGFP+ cells, suggesting that a number of transduced repopulating cells did not express the transgene at that time. Molecular analysis of NOD/SCID BM confirmed the high levels of engraftment of human transduced cells deduced from FACS analysis. Finally, the analysis of the provirus insertion sites by conventional Southern blotting indicated that the human hematopoiesis in the NOD/SCID BM was predominantly oligoclonal.

Efficient transduction of human hematopoietic repopulating cells generating stable engraftment of transgene-expressing cells in NOD/SCID mice

AbstractIn an attempt to develop efficient procedures of human hematopoietic gene therapy, retrovirally transduced CD34+ cord blood cells were transplanted into NOD/SCID mice to evaluate the repopulating potential of transduced grafts. Samples were prestimulated on Retronectin-coated dishes and infected with gibbon ape leukemia virus (GALV)-pseudotyped FMEV vectors encoding the enhanced green fluorescent protein (EGFP). Periodic analyses of bone marrow (BM) from transplanted recipients revealed a sustained engraftment of human hematopoietic cells expressing the EGFP transgene. On average, 33.6% of human CD45+ cells expressed the transgene 90 to120 days after transplantation. Moreover, 11.9% of total NOD/SCID BM consisted of human CD45+ cells expressing the EGFP transgene at this time. The transplantation of purified EGFP+ cells increased the proportion of CD45+ cells positive for EGFP expression to 57.7% at 90 to 120 days after transplantation. At this time, 18.9% and 4.3% of NOD/SCID BM consisted of CD45+/EGFP+ and CD34+/EGFP+ cells, respectively. Interestingly, the transplantation of EGFP− cells purified at 24 hours after infection also generated a significant engraftment of CD45+/EGFP+ and CD34+/EGFP+ cells, suggesting that a number of transduced repopulating cells did not express the transgene at that time. Molecular analysis of NOD/SCID BM confirmed the high levels of engraftment of human transduced cells deduced from FACS analysis. Finally, the analysis of the provirus insertion sites by conventional Southern blotting indicated that the human hematopoiesis in the NOD/SCID BM was predominantly oligoclonal.

Growth Characteristics of Acute Myelogenous Leukemia Progenitors That Initiate Malignant Hematopoiesis in Nonobese Diabetic/Severe Combined Immunodeficient Mice

AbstractThe use of immunodeficient mice, particularly of the nonobese diabetic/severe combined immunodeficient (NOD/SCID) strain, has allowed detection of very primitive malignant progenitors from patients with acute myelogenous leukemia (AML). To define the sensitivity and reproducibility with which the engraftment of different AML cells can be detected, 61 different samples from patients with newly diagnosed AML representing a variety of cytogenetic and French-American-British (FAB) subtypes were injected into NOD/SCID mice. Eight weeks after intravenous injection of 107 AML cells, the average percent of human cells in mouse bone marrow was 13.3%, with 70% of samples showing easily detectable engraftment of CD45+ cells. AML samples with cytogenetic changes associated with a poor clinical prognosis tended to engraft to higher levels than those with changes associated with a good prognosis. Cells with FAB subtypes M3 and, to a lesser extent, M2, engrafted more poorly (P = .002 and .06, respectively) than those from other subtypes. Intraperitoneal injection of human interleukin-3 and Steel factor thrice weekly for 4 weeks did not enhance the levels of AML cell engraftment. However, AML samples that showed cytokine-independent colony growth in methylcellulose assay or expressed growth-factor mRNA in malignant blasts achieved significantly higher levels of engraftment than those which were cytokine dependent in culture or failed to express cytokine message (P < .03 and P < .02, respectively). In 6 patient samples, the frequency of NOD/SCID leukemia-initiating cells (NOD/SL-IC) varied from 0.7 to 45 per 107 cells, which was 200- to 800-fold lower than the frequency of AML long-term culture-initiating cells (AML LTC-IC) in the same samples. Each NOD/SL-IC will produce more than 106leukemic blasts as well as many AML-CFC and AML LTC-IC as detected 8 weeks postinjection into mice. Serial transplant experiments showed the ability of NOD/SL-IC to maintain their own numbers over at least 3 to 4 weeks in vivo. The ability of these progenitors to self-renew combined with their potential to differentiate to produce large numbers of more mature progenitors and leukemic blasts suggests that the NOD/SL-IC assay identifies leukemic ‘stem cells’ that may maintain the malignant clone in human patients. The further use of this assay should facilitate studies of AML stem cell biology and the evolution of novel therapeutic strategies.

Growth Characteristics of Acute Myelogenous Leukemia Progenitors That Initiate Malignant Hematopoiesis in Nonobese Diabetic/Severe Combined Immunodeficient Mice

The use of immunodeficient mice, particularly of the nonobese diabetic/severe combined immunodeficient (NOD/SCID) strain, has allowed detection of very primitive malignant progenitors from patients with acute myelogenous leukemia (AML). To define the sensitivity and reproducibility with which the engraftment of different AML cells can be detected, 61 different samples from patients with newly diagnosed AML representing a variety of cytogenetic and French-American-British (FAB) subtypes were injected into NOD/SCID mice. Eight weeks after intravenous injection of 107 AML cells, the average percent of human cells in mouse bone marrow was 13.3%, with 70% of samples showing easily detectable engraftment of CD45+ cells. AML samples with cytogenetic changes associated with a poor clinical prognosis tended to engraft to higher levels than those with changes associated with a good prognosis. Cells with FAB subtypes M3 and, to a lesser extent, M2, engrafted more poorly (P = .002 and .06, respectively) than those from other subtypes. Intraperitoneal injection of human interleukin-3 and Steel factor thrice weekly for 4 weeks did not enhance the levels of AML cell engraftment. However, AML samples that showed cytokine-independent colony growth in methylcellulose assay or expressed growth-factor mRNA in malignant blasts achieved significantly higher levels of engraftment than those which were cytokine dependent in culture or failed to express cytokine message (P &lt; .03 and P &lt; .02, respectively). In 6 patient samples, the frequency of NOD/SCID leukemia-initiating cells (NOD/SL-IC) varied from 0.7 to 45 per 107 cells, which was 200- to 800-fold lower than the frequency of AML long-term culture-initiating cells (AML LTC-IC) in the same samples. Each NOD/SL-IC will produce more than 106leukemic blasts as well as many AML-CFC and AML LTC-IC as detected 8 weeks postinjection into mice. Serial transplant experiments showed the ability of NOD/SL-IC to maintain their own numbers over at least 3 to 4 weeks in vivo. The ability of these progenitors to self-renew combined with their potential to differentiate to produce large numbers of more mature progenitors and leukemic blasts suggests that the NOD/SL-IC assay identifies leukemic ‘stem cells’ that may maintain the malignant clone in human patients. The further use of this assay should facilitate studies of AML stem cell biology and the evolution of novel therapeutic strategies.