The burden of parainfluenza virus infection in patients with hematological malignancy and hematopoietic stem cell transplant (HSCT) recipients in the absence of active immunization and approved therapy: The role of infection control

Background. Community respiratory viruses, mainly RSV and influenza, are significant causes of morbidity and mortality in patients with leukemia and HSCT recipients. The data on impact of PIV infections in these patients is lacking. Methods. We reviewed the records of patients with leukemia and HSCT recipients who developed PIV infection from Oct'02–Nov'07 to determine the outcome of such infections. Results. We identified 200 patients with PIV infections including 80(40%) patients with leukemia and 120 (60%) recipients of HSCT. Median age was 55 y (17-84 y). As compared to HSCT recipients, patients with leukemia had higher APACHE II score (14 vs. 10, p<0.0001); were more likely to have ANC<500 (48% vs. 10%, p<0.0001) and ALC<200 (45% vs. 23.5%, p=0.02). PIV type III was the commonest isolate (172/200, 86%). Most patients 141/200 (70%) had upper respiratory infection (URI), and 59/200 (30%) had pneumonia at presentation. Patients in leukemia group were more likely to require hospitalization due to PIV infection (77% vs. 36% p=0.0001) and were more likely to progress to pneumonia (61% vs. 39%, p=0.002). Fifty five patients received aerosolized ribavirin and/or IVIG. There were no significant differences in the duration of symptoms, length of hospitalization, progression to pneumonia or mortality between the treated verses untreated group. The clinical outcome was unknown in 13 (6%) patients. Complete resolution of symptoms was noted in 91% (171/187) patients and 9% (16/187) patients died. Mortality rate was 17% (16/95) among patients who had PIV pneumonia, with no significant difference between leukemia and HSCT group (16% vs. 17%). The cause of death was acute respiratory failure and/or multi-organ failure in (13, 81%) patients. Conclusions. Patients with leukemia and HSCT could be at high risk for serious PIV infections including PIV pneumonia. Treatment with aerosolized ribavirin and/or IVIG may not have significant effect on the outcome of PIV infection.^

Respiratory syncytial virus infection in hematopoietic stem cell transplant patients

Respiratory Syncytial Virus (RSV) is a major cause of respiratory tract infections in immunocompromised patients such as children less than 2 years, premature infants with congenital heart disease and chronic lung disease, elderly patients and patients who have undergone hematopoietic stem cell transplant (HSCT). HSCT patients are at high risk of RSV infection, at increased risk of developing pneumonia, and RSV-related mortality. Immunodeficiency can be a major risk factor for severe infection & mortality. Therapy of RSV infection with Ribavirin, Palivizumab and Immunoglobulin has shown to reduce the risk of progression to LRI and mortality, especially if initiated early in the disease. Data on RSV infection in HSCT patients is limited, especially at various levels of immunodeficiency. 323 RSV infections in HSCT patients have been identified between 1/1995 and 8/2009 at University of Texas M D Anderson Cancer Center (UTMDACC). In this proposed study, we attempted to analyze a de-identified database of these cases and describe the epidemiologic characteristics of RSV infection in HSCT patients, the course of the infection, rate of development of pneumonia and RSV-related mortality in HSCT patients at UTMDACC.^ Key words: RSV infections, HSCT patients ^

Inducible caspase 9 suicide gene to improve the safety of allodepleted T cells after haploidentical stem cell transplantation.

Addback of donor T cells following T cell-depleted stem cell transplantation (SCT) can accelerate immune reconstitution and be effective against relapsed malignancy. After haploidentical SCT, a high risk of graft-versus-host disease (GVHD) essentially precludes this option, unless the T cells are first depleted of alloreactive precursor cells. Even then, the risks of severe GVHD remain significant. To increase the safety of the approach and thereby permit administration of larger T cell doses, we used a suicide gene, inducible caspase 9 (iCasp9), to transduce allodepleted T cells, permitting their destruction should administration have adverse effects. We made a retroviral vector encoding iCasp9 and a selectable marker (truncated CD19). Even after allodepletion (using anti-CD25 immunotoxin), donor T cells could be efficiently transduced, expanded, and subsequently enriched by CD19 immunomagnetic selection to >90% purity. These engineered cells retained antiviral specificity and functionality, and contained a subset with regulatory phenotype and function. Activating iCasp9 with a small-molecule dimerizer rapidly produced >90% apoptosis. Although transgene expression was downregulated in quiescent T cells, iCasp9 remained an efficient suicide gene, as expression was rapidly upregulated in activated (alloreactive) T cells. We have demonstrated the clinical feasibility of this approach after haploidentical transplantation by scaling up production using clinical grade materials.

IDENTIFICATION AND CHARACTERIZATION OF DISTINCT POPULATIONS OF CLONOGENIC BONE MARROW STROMAL CELLS CAPABLE OF TRANSFERRING THE HEMATOPOIETIC MICROENVIRONMENT IN VIVO AND SUPPORTING LT-HSCs IN VITRO

Bone marrow (BM) stromal cells are ascribed two key functions, 1) stem cells for non-hematopoietic tissues (MSC) and 2) as components of the hematopoietic stem cell niche. Current approaches studying the stromal cell system in the mouse are complicated by the low yield of clonogenic progenitors (CFU-F). Given the perivascular location of MSC in BM, we developed an alternative methodology to isolate MSC from mBM. An intact ‘plug’ of bone marrow is expelled from bones and enzymatically disaggregated to yield a single cell suspension. The recovery of CFU-F (1917.95+199) reproducibly exceeds that obtained using the standard BM flushing technique (14.32+1.9) by at least 2 orders of magnitude (P<0.001; N = 8) with an accompanying 196-fold enrichment of CFU-F frequency. Purified BM stromal and vascular endothelial cell populations are readily obtained by FACS. A detailed immunophenotypic analysis of lineage depleted BM identified PDGFRαβPOS stromal cell subpopulations distinguished by their expression of CD105. Both subpopulations retained their original phenotype of CD105 expression in culture and demonstrate MSC properties of multi-lineage differentiation and the ability to transfer the hematopoietic microenvironment in vivo. To determine the capacity of either subpopulation to support long-term multi-lineage reconstituting HSCs, we fractionated BM stromal cells into either the LinNEGPDGFRαβPOSCD105POS and LINNEGPDGFRαβPOSCD105LOW/- populations and tested their capacity to support LT-HSC by co-culturing each population with either 1 or 10 HSCs for 10 days. Following the 10 day co-culture period, both populations supported transplantable HSCs from 10 cells/well co-cultures demonstrating high levels of donor repopulation with an average of 65+23.6% chimerism from CD105POS co-cultures and 49.3+19.5% chimerism from the CD105NEG co-cultures. However, we observed a significant difference when mice were transplanted with the progeny of a single co-cultured HSC. In these experiments, CD105POS co-cultures (100%) demonstrated long-term multi- lineage reconstitution, while only 4 of 8 mice (50%) from CD105NEG -single HSC co-cultures demonstrated long-term reconstitution, suggesting a more limited expansion of functional stem cells. Taken together, these results demonstrate that the PDGFRαβCD105POS stromal cell subpopulation is distinguished by a unique capacity to support the expansion of long-term reconstituting HSCs in vitro.

Intravenous mesenchymal stem cell therapy for traumatic brain injury.

OBJECT: Cell therapy has shown preclinical promise in the treatment of many diseases, and its application is being translated to the clinical arena. Intravenous mesenchymal stem cell (MSC) therapy has been shown to improve functional recovery after traumatic brain injury (TBI). Herein, the authors report on their attempts to reproduce such observations, including detailed characterizations of the MSC population, non-bromodeoxyuridine-based cell labeling, macroscopic and microscopic cell tracking, quantification of cells traversing the pulmonary microvasculature, and well-validated measurement of motor and cognitive function recovery. METHODS: Rat MSCs were isolated, expanded in vitro, immunophenotyped, and labeled. Four million MSCs were intravenously infused into Sprague-Dawley rats 24 hours after receiving a moderate, unilateral controlled cortical impact TBI. Infrared macroscopic cell tracking was used to identify cell distribution. Immunohistochemical analysis of brain and lung tissues 48 hours and 2 weeks postinfusion revealed transplanted cells in these locations, and these cells were quantified. Intraarterial blood sampling and flow cytometry were used to quantify the number of transplanted cells reaching the arterial circulation. Motor and cognitive behavioral testing was performed to evaluate functional recovery. RESULTS: At 48 hours post-MSC infusion, the majority of cells were localized to the lungs. Between 1.5 and 3.7% of the infused cells were estimated to traverse the lungs and reach the arterial circulation, 0.295% reached the carotid artery, and a very small percentage reached the cerebral parenchyma (0.0005%) and remained there. Almost no cells were identified in the brain tissue at 2 weeks postinfusion. No motor or cognitive functional improvements in recovery were identified. CONCLUSIONS: The intravenous infusion of MSCs appeared neither to result in significant acute or prolonged cerebral engraftment of cells nor to modify the recovery of motor or cognitive function. Less than 4% of the infused cells were likely to traverse the pulmonary microvasculature and reach the arterial circulation, a phenomenon termed the "pulmonary first-pass effect," which may limit the efficacy of this therapeutic approach. The data in this study contradict the findings of previous reports and highlight the potential shortcomings of acute, single-dose, intravenous MSC therapy for TBI.

Transplantation of human embryonic stem cell-derived alveolar epithelial type II cells abrogates acute lung injury in mice.

Respiratory diseases are a major cause of mortality and morbidity worldwide. Current treatments offer no prospect of cure or disease reversal. Transplantation of pulmonary progenitor cells derived from human embryonic stem cells (hESCs) may provide a novel approach to regenerate endogenous lung cells destroyed by injury and disease. Here, we examine the therapeutic potential of alveolar type II epithelial cells derived from hESCs (hES-ATIICs) in a mouse model of acute lung injury. When transplanted into lungs of mice subjected to bleomycin (BLM)-induced acute lung injury, hES-ATIICs behaved as normal primary ATIICs, differentiating into cells expressing phenotypic markers of alveolar type I epithelial cells. Without experiencing tumorigenic side effects, lung injury was abrogated in mice transplanted with hES-ATIICs, demonstrated by recovery of body weight and arterial blood oxygen saturation, decreased collagen deposition, and increased survival. Therefore, transplantation of hES-ATIICs shows promise as an effective therapeutic to treat acute lung injury.

Subacute neural stem cell therapy for traumatic brain injury.

INTRODUCTION: Traumatic brain injury (TBI) frequently results in devastating and prolonged morbidity. Cellular therapy is a burgeoning field of experimental treatment that has shown promise in the management of many diseases, including TBI. Previous work suggests that certain stem and progenitor cell populations migrate to sites of inflammation and improve functional outcome in rodents after neural injury. Unfortunately, recent study has revealed potential limitations of acute and intravenous stem cell therapy. We studied subacute, direct intracerebral neural stem and progenitor cell (NSC) therapy for TBI. MATERIALS AND METHODS: The NSCs were characterized by flow cytometry and placed (400,000 cells in 50 muL 1x phosphate-buffered saline) into and around the direct injury area, using stereotactic guidance, of female Sprague Dawley rats 1 wk after undergoing a controlled cortical impact injury. Immunohistochemistry was used to identify cells located in the brain at 48 h and 2 wk after administration. Motor function was assessed using the neurological severity score, foot fault, rotarod, and beam balance. Cognitive function was assessed using the Morris water maze learning paradigm. Repeated measures analysis of variance with post-hoc analysis were used to determine significance at P < 0.05. RESULTS: Immunohistochemistry analysis revealed that 1.4-1.9% of infused cells remained in the neural tissue at 48 h and 2 wk post placement. Nearly all cells were located along injection tracks at 48 h. At 2 wk some cell dispersion was apparent. Rotarod motor testing revealed significant increases in maximal speed among NSC-treated rats compared with saline controls at d 4 (36.4 versus 27.1 rpm, P < 0.05) and 5 (35.8 versus 28.9 rpm, P < 0.05). All other motor and cognitive evaluations were not significantly different compared to controls. CONCLUSIONS: Placement of NSCs led to the cells incorporating and remaining in the tissues 2 wk after placement. Motor function tests revealed improvements in the ability to run on a rotating rod; however, other motor and cognitive functions were not significantly improved by NSC therapy. Further examination of a dose response and optimization of placement strategy may improve long-term cell survival and maximize functional recovery.

Expression of the neural stem cell markers NG2 and L1 in human angiomyolipoma: are angiomyolipomas neoplasms of stem cells?

Angiomyolipomas are benign tumors of the kidney which express phenotypes of smooth muscle, fat, and melanocytes. These tumors appear with increased frequency in the autosomal dominant disorder tuberous sclerosis and are the leading cause of morbidity in adults with tuberous sclerosis. While benign, these tumors are capable of provoking life threatening hemorrhage and replacement of the kidney parenchyma, resulting in renal failure. The histogenesis of these tumors is currently unclear, although currently, we believe these tumors arise from "perivascular epithelioid cells" of which no normal counterpart has been convincingly demonstrated. Recently, stem cell precursors have been recognized that can give rise to smooth muscle and melanocytes. These precursors have been shown to express the neural stem cell marker NG2 and L1. In order to determine whether angiomyolipomas, which exhibit smooth muscle and melanocytic phenotypes, express NG2 and L1, we performed immunocytochemistry on a cell line derived from a human angiomyolipoma, and found that these cells are uniformly positive. Immunohistochemistry of human angiomyolipoma specimens revealed uniform staining of tumor cells, while renal cell carcinomas revealed positivity only of angiogenic vessels. These results support a novel histogenesis of angiomyolipoma as a defect in differentiation of stem cell precursors.

Current United States stem cell policy considerations and a recommendation for an alternative policy based on guidelines from existing international policies

Embryonic stem cell research is a widely debated topic in modern politics and religion. Differing views on the fetal rights conflict with the rights of an embryo. Those who believe an embryo has the same human qualities as a fetus accordingly believe embryonic stem cell research is unethical because it destroys a potential human life. However, scientists advocate the embryo does not have human qualities and should be used for valuable research in the stem cell field. Stem cell research may lead to vast developments in medical treatments, including cancer and brain conditions and injuries that are currently incurable. ^ The current stem cell policy introduced by President Bush in 2001 in an attempt to balance the moral issues with the need for scientific research has broad negative implications on the furthering of stem cell research. There is a limited diversity of available stem cell lines, there may be constitutional issues, there is an increasing disparity between the public and private research spheres, and the U.S. is struggling to maintain its scientific community. The U.S. must develop a new stem cell research policy that will balance the interest of science and public health with the moral issues that concern the public. ^ The United Kingdom allows researchers great liberty in conducting research, permitting the creation of embryos for the sole purpose of research, while Germany is equally conservative in their laws, as their policies support the philosophy that all embryos deserve the protection of full life. The United States should adopt a policy that takes the "middle ground" approach and permit research on excess embryos created for IVF purposes, rather than simply discarding those potentially valuable research tools. ^

Federal regulation of human embryonic stem cell research

President George W. Bush's 2001 statement, which laid out guidelines for research that uses human embryonic stem cells to qualify for federal funding, intends to prevent new embryonic stem cell lines from being developed, by prohibiting the federal funding of research that uses embryonic stem cell lines other than those that existed at the time of the policy's inception and were approved by the National Institutes of Health. This policy raises questions of medical and technological ethics and the governments' role in making decisions regarding the advancement of science based on moral and political opinions. Federal stem cell usage policy directly affects scientific research efforts that are currently on the path to understanding the mechanisms of cell differentiation and could potentially offer answers and therapies for disabilities and many chronic diseases. By reviewing the current literature on the background information on human embryonic stem cells, including what they are, where they come from, how they are used for research purposes, and the ethical controversy surrounding their use, I have researched and reported the impact of the 2001 policy on medical research. ^ Both those who support the current policy on human embryonic stem cell research and those who are advocates for policy change have relevant arguments and varying opinions on human embryonic stem cell usage itself. The ethical implication of how embryonic stem cells are obtained has led to fierce debate. This paper presents many arguments for and against hESC research in addition to the policy governing their use. This analysis concludes that the current policy on federal funding of human embryonic stem cell research should be revised to allow research using new stem lines to be eligible for federal funding under specific guidelines. Supporting evidence for this recommendation is provided.^

Children with acute leukemia: A comparison of outcomes and cost-effectiveness from allogeneic blood stem cell and bone marrow transplantation

The relative merits of PBSCT versus BMT for children with standard and high risk hematologic malignancies remain unclear. In a retrospective single center study, we compared allogeneic peripheral blood stem cell transplantation (PBSCT) (n=30) with bone marrow transplantation (BMT) (n=110) in children with acute leukemia. We studied recipients of HLA matched sibling stem cells, and of stem cells from alternative donors (HLA mismatched and/or unrelated) and determined whether sourcing the stem cells from PB or marrow affected engraftment, incidence of acute and chronic GvHD, and disease-free survival at 1 year. Our results show a modest reduction in time to engraftment from PB stem cells and no greater risk of GvHD, but illustrate that the severity of the underlying disease is by far the greatest determinant of 1 year survival. Patients in the BMT group had a higher treatment success rate and lower costs than the recipients of the PBSCT within the standard but not the high risk disease group, where the treatment success rate and the cumulative costs were lower in the PBSCT group compared to the BMT group. Our current incremental cost-effectiveness ratio and analysis of uncertainty suggest that allogeneic transplantation of bone marrow grafts was a more cost-effective treatment option compared to peripheral blood stem cells in patients with standard risk childhood acute leukemia disease. For high risk disease our data are less prescriptive, since the differences were more limited and the range of costs much larger. Neither option demonstrated a clear advantage from a cost-effectiveness standpoint.^

Elucidating the role of mesenchymal stem cell participation in the tumor microenvironment

To meet the requirements for rapid tumor growth, a complex array of non-neoplastic vascular, fibroblastic, and immune cells are recruited to the tumor microenvironment. Understanding the origin, composition, and mechanism(s) for recruitment of these stromal components will help identify areas for therapeutic intervention. Previous findings have suggested that ex-vivo expanded bone marrow-derived MSC home to the sites of tumor development, responding to inflammatory signals and can serve as effective drug delivery vehicles. Therefore, we first sought to fully assess conditions under which MSC migrate to and incorporate into inflammatory microenvironments and the consequences of modulated inflammation. MSC delivered to animals bearing inflammatory insults were monitored by bioluminescence imaging and displayed specific tropism and selective incorporation into all tumor and wound sites. These findings were consistent across routes of tumor establishment, MSC administration, and immunocompetence. MSC were then used as drug delivery vehicles, transporting Interferon β to sites of pancreatic tumors. This therapy was effective at inhibiting pancreatic tumor growth under homeostatic conditions, but inhibition was lost when inflammation was decreased with CDDO-Me combination treatment. Next, to examine the endogenous tumor microenvironment, a series of tissue transplant experiments were carried out in which tissues were genetically labeled and engrafted in recipients prior to tumor establishment. Tumors were then analyzed for markers of tumor associated fibroblasts (TAF): α-smooth muscle actin (α-SMA), nerve glia antigen 2 (NG2), fibroblast activation protein (FAP), and fibroblast specific protein (FSP) as well as endothelial marker CD31 and macrophage marker F4/80. We determined the majority of α-SMA+, NG2+ and CD31+ cells were non-bone marrow derived, while most FAP+, FSP+, and F4/80+ cells were recruited from the bone marrow. In accord, transplants of prospectively isolated BM MSC prior to tumor development indicated that these cells were recruited to the tumor microenvironment and co-expressed FAP and FSP. In contrast, fat transplant experiments revealed recruited fat derived cells co-expressed α-SMA, NG2, and CD31. These results indicate TAF are a heterogeneous population composed of subpopulations with distinct tissues of origin. These models have provided a platform upon which further investigation into tumor microenvironment composition and tests for candidate drugs can be performed. ^

LDOC1, A NOVEL BIOMARKER OF PROGNOSIS IN CHRONIC LYMPHOCYTIC LEUKEMIA

In chronic lymphocytic leukemia (CLL), one of the best predictors of outcome is the somatic mutation status of the immunoglobulin heavy chain variable region (IGHV) genes. Patients whose CLL cells have unmutated IGHV genes have a median survival of 8 years; those with mutated IGHV genes have a median survival of 25 years. To identify new prognostic biomarkers and molecular targets for therapy in untreated CLL patients, we reanalyzed the raw data from four published gene expression profiling microarray studies. Of 88 candidate biomarkers associated with IGHV somatic mutation status, we identified LDOC1 (Leucine Zipper, Down-regulated in Cancer 1), as one of the most significantly differentially expressed genes that distinguished mutated from unmutated CLL cases. LDOC1 is a putative transcription factor of unknown function in B-cell development and CLL pathophysiology. Using a highly sensitive quantitative RT-PCR (QRT-PCR) assay, we confirmed that LDOC1 mRNA was dramatically down-regulated in mutated compared to unmutated CLL cases. Expression of LDOC1 mRNA was also vii strongly associated with other markers of poor prognosis, including ZAP70 protein and cytogenetic abnormalities of poor prognosis (deletions of chromosomes 6q21, 11q23, and 17p13.1, and trisomy 12). CLL cases positive for LDOC1 mRNA had significantly shorter overall survival than negative cases. Moreover, in a multivariate model, LDOC1 mRNA expression predicted overall survival better than IGHV mutation status or ZAP70 protein, among the best markers of prognosis in CLL. We also discovered LDOC1S, a new LDOC1 splice variant. Using isoform-specific QRT-PCR assays that we developed, we found that both isoforms were expressed in normal B cells (naïve > memory), unmutated CLL cells, and in B-cell non-Hodgkin lymphomas with unmutated IGHV genes. To investigate pathways in which LDOC1 is involved, we knocked down LDOC1 in HeLa cells and performed global gene expression profiling. GFI1 (Growth Factor-Independent 1) emerged as a significantly up-regulated gene in both HeLa cells and CLL cells that expressed high levels of LDOC1. GFI1 oncoprotein is implicated in hematopoietic stem cell maintenance, lymphocyte development, and lymphomagenesis. Our findings indicate that LDOC1 mRNA is an excellent biomarker of overall survival in CLL, and may contribute to B-cell differentiation and malignant transformation.

THE CONSEQUENCES OF DISRUPTING THE MDM2-P53 BALANCE IN HEMATOPOIESIS

The bone marrow accommodates hematopoietic stem cells and progenitors. These cells provide an indispensible resource for replenishing the blood constituents throughout an organism’s life. A tissue with such a high turn-over rate mandates intact cycling checkpoint and apoptotic pathways to avoid inappropriate cell proliferation and ultimately the development of leukemias. p53, a major tumor suppressor, is a transcription factor that regulates cell cycle, and induces apoptosis and senescence. Mice inheriting a hypomorphic p53 allele in the absence of Mdm2, a p53 inhibitor, have elevated p53 cell cycle activity and die by postnatal day 13 due to hematopoietic failure. Hematopoiesis progresses normally during embryogenesis until it moves to the bone marrow in late development. Increased oxidative stress in the bone marrow compartment postnatally is the impediment for normal hematopoiesis via activation of p53. p53 in turn stimulates the generation of more reactive oxygen species and depletes bone marrow cellularity. Also, p53 exerts various defects on the hematopoietic niche by increasing mesenchymal lineage populations and their differentiation. Hematopoietic defects are rescued with antioxidants or when cells are cultured at low oxygen levels. Deletion of p16 partially rescues bone marrow cellularity and progenitors via a p53-independent pathway. Thus, although p53 is required to inhibit tumorigenesis, Mdm2 is required to control ROS-induced p53 levels for sustainable hematopoiesis and survival during homeostasis.

Rescue from apoptosis in early (CD34 selected) versus late (CD34 unselected) human hematopoietic cells by VLA-4 and VCAM-1 dependent adhesion to bone marrow stromal cells

The interaction of hematopoietic precursor cell with bone marrow stromal cells is assumed to be important to the survival of hematopoietic precursor cells during hematopoietic cell long-term culture. Early hematopoietic stem cells are preferentially found within the stromal adherent cell fraction in primary long-term bone marrow cultures. The purpose of this dissertation was to understand the molecular mechanisms that govern these interactions for the regulation of survival and proliferation of early versus late hematopoietic cells.^ Monoclonal antibodies to the VLA-4 recognize the alpha4 beta1 integrin receptor on human hematopoietic cells. This monoclonal antibody blocks the adhesion between early hematopoietic progenitor cells (CD34 selected cells) and stromal cells when added to cultures of these cells. Addition of the VLA-4 monoclonal antibody to cultures of stromal cells and CD34 selected cells was shown to induce apoptosis of CD34 selected cells in these CD34 selected cell/stromal cell cocultures, as measured by the terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick end-labeling method. In contrast to these experiments with early hematopoietic progenitor cells (CD34+), the level of adhesion between more differentiated cells (unfractionated hematopoietic cells) and stromal cells was not significantly altered by addition of the anti-VLA-4 monoclonal antibody. Similarly, the level of apoptosis of unfractionated hematopoietic cells was not significantly increased by the addition of anti-VLA-4 monoclonal antibody to cultures of the latter cells with stromal cells. The binding of the unfractionated cells is less than that of the CD34 selected. Since there is no difference between the alpha4 beta1 integrin expression level of the early and late myeloid cells, there may be a difference in the functional state of the integrin between the early and late myeloid cells. We also show that CD34+ selected precursor cells proliferate at a higher rate when these cells are plated on recombinant VCAM-1 molecules. These data indicate that the alpha4beta1 integrin receptor (VLA-4) plays a central role in the apoptosis rescue function which results from the anchorage-dependent growth of the CD34 selected early hematopoietic cells on stromal cells. The data suggest that these apoptosis rescue pathways have less significance as the cells mature and become anchorage-independent in their growth. These data should assist in the design of systems for the ex vivo proliferation and transduction of early hematopoietic cells for genetic therapy. ^