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Human CD34  +  stem cells are mobilized from bone marrow to sites of tissue ischemia and play an important role in tissue revascularization. This study used a murine model to test the hypothesis that intravitreal injection of human CD34  +  stem cells harvested from bone marrow (BMSCs) can have protective effects in eyes with diabetic retinopathy. Streptozotocin-induced diabetic mice (C57BL/6J) were used as a model for diabetic retinopathy. Subcutaneous implantation of Alzet pump, loaded with Tacrolimus and Rapamycin, 5 days prior to intravitreal injection provided continuous systemic immunosuppression for the study duration to avoid rejection of human cells. Human CD34  +  BMSCs were harvested from the mononuclear cell fraction of bone marrow from a healthy donor using magnetic beads. The CD34  +  cells were labeled with enhanced green fluorescent protein (EGFP) using a lentiviral vector. The right eye of each mouse received an intravitreal injection of 50,000 EGFP-labeled CD34  +  BMSCs or phosphate buffered saline (PBS). Simultaneous multimodal in vivo retinal imaging system consisting of fluorescent scanning laser ophthalmoscopy (enabling fluorescein angiography), optical coherence tomography (OCT) and OCT angiography was used to confirm the development of diabetic retinopathy and study the in vivo migration of the EGFP-labeled CD34  +  BMSCs in the vitreous and retina following intravitreal injection. After imaging, the mice were euthanized, and the eyes were removed for immunohistochemistry. In addition, microarray analysis of the retina and retinal flat mount analysis of retinal vasculature were performed. The development of retinal microvascular changes consistent with diabetic retinopathy was visualized using fluorescein angiography and OCT angiography between 5 and 6 months after induction of diabetes in all diabetic mice. These retinal microvascular changes include areas of capillary nonperfusion and late leakage of fluorescein dye. Multimodal in vivo imaging and immunohistochemistry identified EGFP-labeled cells in the superficial retina and along retinal vasculature at 1 and 4 weeks following intravitreal cell injection. Microarray analysis showed changes in expression of 162 murine retinal genes following intravitreal CD34  +  BMSC injection when compared to PBS-injected control. The major molecular pathways affected by intravitreal CD34  +  BMSC injection in the murine retina included pathways implicated in the pathogenesis of diabetic retinopathy including Toll-like receptor, MAP kinase, oxidative stress, cellular development, assembly and organization pathways. At 4 weeks following intravitreal injection, retinal flat mount analysis showed preservation of the retinal vasculature in eyes injected with CD34  +  BMSCs when compared to PBS-injected control. The study findings support the hypothesis that intravitreal injection of human CD34  +  BMSCs results in retinal homing and integration of these human cells with preservation of the retinal vasculature in murine eyes with diabetic retinopathy., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Purpose: Intravitreal murine lineage-negative bone marrow (BM) hematopoietic cells slow down retinal degeneration. Because human BM CD34+ hematopoietic cells are not precisely comparable to murine cells, this study examined the effect of intravitreal human BM CD34+ cells on the degenerating retina using a murine model., Methods: C3H/HeJrd1/rd1 mice, immunosuppressed systemically with tacrolimus and rapamycin, were injected intravitreally with PBS (n = 16) or CD34+ cells (n = 16) isolated from human BM using a magnetic cell sorter and labeled with enhanced green fluorescent protein (EGFP). After 1 and 4 weeks, the injected eyes were imaged with scanning laser ophthalmoscopy (SLO)/optical coherence tomography (OCT) and tested with electroretinography (ERG). Eyes were harvested after euthanasia for immunohistochemical and microarray analysis of the retina., Results: In vivo SLO fundus imaging visualized EGFP-labeled cells within the eyes following intravitreal injection. Simultaneous OCT analysis localized the EGFP-labeled cells on the retinal surface resulting in a saw-toothed appearance. Immunohistochemical analysis of the retina identified EGFP-labeled cells on the retinal surface and adjacent to ganglion cells. Electroretinography testing showed a flat signal both at 1 and 4 weeks following injection in all eyes. Microarray analysis of the retina following cell injection showed altered expression of more than 300 mouse genes, predominantly those regulating photoreceptor function and maintenance and apoptosis., Conclusions: Intravitreal human BM CD34+ cells rapidly home to the degenerating retinal surface. Although a functional benefit of this cell therapy was not seen on ERG in this rapidly progressive retinal degeneration model, molecular changes in the retina associated with CD34+ cell therapy suggest potential trophic regenerative effects that warrant further exploration.

The successful suppression of human immunodeficiency virus (HIV) in the "Berlin Patient" has highlighted the ability of HIV-resistant hematopoietic stem cells to offer a potential functional cure for HIV-infected patients. HIV stem cell gene therapy can mimic this result by genetically modifying a patient's own cells with anti-HIV genes. Previous attempts of HIV gene therapy have been hampered by a low percentage of transplanted HIV-resistant cells which has led to minimal clinical efficacy. In our current study, we have evaluated the in vitro and in vivo safety and efficacy of a truncated/mutated form of human CD25 preselective anti-HIV lentiviral vector in human hematopoietic stem cells. This preselective vector allows us to purify vector-transduced cells prior to transplantation so an increased percentage of gene-modified cells can be delivered. Here, we demonstrate the safety of this strategy with successful engraftment and multilineage hematopoiesis of transduced cells in a humanized NOD-RAG1-/-IL-2rγ-/- knockout mouse model. Efficacy was also demonstrated with significant protection from HIV-1 infection including maintenance of human CD4+ cell levels and a decrease in HIV-1 plasma viremia. Collectively, these results establish the utility of this HIV stem cell gene therapy strategy and bring it closer to providing a functional cure for HIV-infected patients., (© 2014 AlphaMed Press.)

HIV gene therapy has the potential to offer an alternative to the use of current small-molecule antiretroviral drugs as a treatment strategy for HIV-infected individuals. Therapies designed to administer HIV-resistant stem cells to an infected patient may also provide a functional cure, as observed in a bone marrow transplant performed with hematopoietic stem cells (HSCs) homozygous for the CCR5-Δ32-bp allele. In our current studies, preclinical evaluation of a combination anti-HIV lentiviral vector was performed, in vivo, in humanized NOD-RAG1(-/-) IL2rγ(-/-) knockout mice. This combination vector, which displays strong preintegration inhibition of HIV-1 infection in vitro, contains a human/rhesus macaque TRIM5α isoform, a CCR5 short hairpin RNA (shRNA), and a TAR decoy. Multilineage hematopoiesis from anti-HIV lentiviral vector-transduced human CD34(+) HSCs was observed in the peripheral blood and in various lymphoid organs, including the thymus, spleen, and bone marrow, of engrafted mice. Anti-HIV vector-transduced CD34(+) cells displayed normal development of immune cells, including T cells, B cells, and macrophages. The anti-HIV vector-transduced cells also displayed knockdown of cell surface CCR5 due to the expression of the CCR5 shRNA. After in vivo challenge with either an R5-tropic BaL-1 or X4-tropic NL4-3 strain of HIV-1, maintenance of human CD4(+) cell levels and a selective survival advantage of anti-HIV gene-modified cells were observed in engrafted mice. The data provided from our study confirm the safety and efficacy of this combination anti-HIV lentiviral vector in a hematopoietic stem cell gene therapy setting for HIV and validates its potential application in future clinical trials.

Purpose: To determine long-term safety of intravitreal administration of good manufacturing practice (GMP)-grade human bone-marrow-derived CD34(+) cells in NOD-SCID (nonobese diabetic-severe combined immunodeficiency) mice with acute retinal ischemia-reperfusion injury, a model for retinal vasculopathy., Method: Acute ischemia-reperfusion injury was induced in the right eye of adult NOD-SCID mice (n = 23) by transient elevation of intraocular pressure. Seven days later, 12 injured eyes and 5 normal contralateral eyes were injected each intravitreally with 5 × 10(4) CD34(+) cells isolated under GMP conditions from a healthy human donor bone marrow using an immunomagnetic cell isolation system. The remaining 11 injured eyes were not treated and served as controls. Mice were euthanized 1 day, 4 months, and 8 months later. Both eyes were enucleated and examined by immunohistochemical analysis and hematoxylin and eosin staining. Among mice followed for 8 months, electroretinography (ERG) was performed on both eyes before euthanization. All major organs were examined grossly and histologically after serial sectioning., Results: Immunohistochemical staining 4 months after injection showed detectable CD34(+) cells in the retinal vasculature. ERG at 8 months after CD34(+) cell injection showed signals that were similar in untreated eyes. Histology of the enucleated eyes injected with CD34(+) cells showed no intraocular tumor or abnormal tissue growth after 8 months. Histologic analysis of all major organs showed no abnormal proliferation of human cells., Conclusions: Intravitreal administration of GMP-grade human bone-marrow-derived CD34(+) cells appears to be well tolerated long-term in eyes with acute retinal ischemic injury. A clinical trial will start to further explore this therapy.

Induced pluripotent stem cells (iPSCs) have radically advanced the field of regenerative medicine by making possible the production of patient-specific pluripotent stem cells from adult individuals. By developing iPSCs to treat HIV, there is the potential for generating a continuous supply of therapeutic cells for transplantation into HIV-infected patients. In this study, we have used human hematopoietic stem cells (HSCs) to generate anti-HIV gene expressing iPSCs for HIV gene therapy. HSCs were dedifferentiated into continuously growing iPSC lines with four reprogramming factors and a combination anti-HIV lentiviral vector containing a CCR5 short hairpin RNA (shRNA) and a human/rhesus chimeric TRIM5α gene. Upon directed differentiation of the anti-HIV iPSCs toward the hematopoietic lineage, a robust quantity of colony-forming CD133(+) HSCs were obtained. These cells were further differentiated into functional end-stage macrophages which displayed a normal phenotypic profile. Upon viral challenge, the anti-HIV iPSC-derived macrophages exhibited strong protection from HIV-1 infection. Here, we demonstrate the ability of iPSCs to develop into HIV-1 resistant immune cells and highlight the potential use of iPSCs for HIV gene and cellular therapies.

The use of nanometer-sized iron oxide particles combined with molecular imaging techniques enables dynamic studies of homing and trafficking of human hematopoietic stem cells (HSC). Identifying clinically applicable strategies for loading nanoparticles into primitive HSC requires strictly defined culture conditions to maintain viability without inducing terminal differentiation. In the current study, fluorescent molecules were covalently linked to dextran-coated iron oxide nanoparticles (Feridex) to characterize human HSC labeling to monitor the engraftment process. Conjugating fluorophores to the dextran coat for fluorescence-activated cell sorting purification eliminated spurious signals from nonsequestered nanoparticle contaminants. A short-term defined incubation strategy was developed that allowed efficient labeling of both quiescent and cycling HSC, with no discernable toxicity in vitro or in vivo. Transplantation of purified primary human cord blood lineage-depleted and CD34(+) cells into immunodeficient mice allowed detection of labeled human HSC in the recipient bones. Flow cytometry was used to precisely quantitate the cell populations that had sequestered the nanoparticles and to follow their fate post-transplantation. Flow cytometry endpoint analysis confirmed the presence of nanoparticle-labeled human stem cells in the marrow. The use of fluorophore-labeled iron oxide nanoparticles for fluorescence imaging in combination with flow cytometry allows evaluation of labeling efficiencies and homing capabilities of defined human HSC subsets.

This unit provides methods for introducing genes into human hematopoietic progenitor cells. The Basic Protocol describes isolation of CD34(+) cells, transduction of these cells with a retroviral vector on fibronectin-coated plates, assaying the efficiency of transduction, and establishing long-term cultures. Support protocols describe methods for maintenance of vector-producing fibroblasts (VPF) and supernatant collection from these cells, screening medium components for the ability to support hematopoietic cell growth, and establishing colonies from long-term cultures. Other protocols provide PCR-based methods to analyze individual colonies for transduction, methods to analyze cells harvested from long-term cultures, and procedures for freezing and thawing of hematopoietic cells., (Copyright 2008 by John Wiley & Sons, Inc.)

Within the bone marrow (BM), hematopoietic progenitor cells (HPCs) are localized in poorly oxygenated niches where they interact with the surrounding osteoblasts (OBs) through VLA4/VCAM-1 engagement, and are exposed to interleukin-6 (IL-6), stem cell factor (SCF), and chemokines such as CXCL12 (OB factors). Umbilical cord (UC) is more highly oxygenated that the BM microenvironment. When UC-HPCs are exposed to the 2% to 3% O(2) concentration found in the bone endosteum, their survival is significantly decreased. However, engagement of VLA-4 integrins on UCB-derived CD34(+) cells reduced cell death in 2% to 3% O(2) conditions, which was associated with an increase in phospho-Ser473 AKT and an increase in phospho-Ser9 GSK3b. Consistent with the role of GSK3b in destabilizing beta-catenin, there was more cytoplasmic beta-catenin in UC-HPCs exposed to 2% to 3% O(2) on fibronectin, compared with suspension culture. UC-HPCs cultured at 2% to 3% O(2) with OB factors showed an increase in nuclear beta-catenin and persistence of a small pool of CD34(+)38(-) HPCs. CFU assays followed by surface phenotyping of the plated colonies showed improved maintenance of mixed lineage colonies with both erythroid and megakaryocytic precursors. These studies provide a biologic perspective for how UC-derived HPCs adapt to the bone endosteum, which is low in oxygen and densely populated by osteoblasts.

MRI has been employed to elucidate the migratory behavior of stem/progenitor cells noninvasively in vivo with traditional proton (1H) imaging of iron oxide nanoparticle-labeled cells. Alternatively, we demonstrate that fluorine (19F) MRI of cells labeled with different types of liquid perfluorocarbon (PFC) nanoparticles produces unique and sensitive cell markers distinct from any tissue background signal. To define the utility for cell tracking, mononuclear cells harvested from human umbilical cord blood were grown under proendothelial conditions and labeled with nanoparticles composed of two distinct PFC cores (perfluorooctylbromide and perfluoro-15-crown-5 ether). The sensitivity for detecting and imaging labeled cells was defined on 11.7T (research) and 1.5T (clinical) scanners. Stem/progenitor cells (CD34+ CD133+ CD31+) readily internalized PFC nanoparticles without aid of adjunctive labeling techniques, and cells remained functional in vivo. PFC-labeled cells exhibited distinct 19F signals and were readily detected after both local and intravenous injection. PFC nanoparticles provide an unequivocal and unique signature for stem/progenitor cells, enable spatial cell localization with 19F MRI, and permit quantification and detection of multiple fluorine signatures via 19F MR spectroscopy. This method should facilitate longitudinal investigation of cellular events in vivo for multiple cell types simultaneously.

We have previously shown that engagement of the integrins VLA-4 and VLA-5 to the fibronectin fragment CH-296 in combination with cytokines sustained the capacity of cultured human CD34(+) cells to undergo hematopoiesis in immunodeficient mice for 7 to 12 months, whereas this capacity was rapidly lost in cells cultured in suspension with the same cytokines. In the current study, we assessed the molecular pathways that might explain the loss of long-term engraftment capacity in cells cultured in suspension. Although the cell cycle profile was similar between cells cultured in suspension versus on fibronectin, levels of cell death were higher in the suspended cultures. While the CDK inhibitors p27Kip1 and p57Kip2 were present at equal levels in cells from both cultures, low levels of p21Cip1 were detectable only in the cytoplasmic compartment of cells cultured in suspension. Cytoplasmic location of p21Cip1 has been linked to monocytic differentiation. The levels of c-myb and GATA-2, transcription factors associated with stem cell maintenance, were higher in cells cultured on fibronectin as compared with suspension. In contrast, the levels of PU.1, which is induced during myeloid differentiation, were higher in cells cultured in suspension. There were no significant differences in surface expression of CD34 on the cells after culture, but total CD34 protein, assessed by immunoblotting, was significantly higher in cells cultured on fibronectin. Our data suggest that, in the presence of cytokines, the engagement of VLA-4 and VLA-5 integrins to the fibronectin fragment CH-296 preserves the expression of specific transcription factors associated with primitive stem cell maintenance. In contrast, a lack of integrin engagement leads to the induction of cellular markers associated with myeloid differentiation.

The development of novel cell-based therapies requires understanding of distinct human hematopoietic stem and progenitor cell populations. We recently isolated reconstituting hematopoietic stem cells (HSCs) by lineage depletion and purification based on high aldehyde dehydrogenase activity (ALDH(hi)Lin- cells). Here, we further dissected the ALDH(hi)-Lin- population by selection for CD133, a surface molecule expressed on progenitors from hematopoietic, endothelial, and neural lineages. ALDH(hi)CD133+Lin- cells were primarily CD34+, but also included CD34-CD38-CD133+ cells, a phenotype previously associated with repopulating function. Both ALDH(hi)CD133-Lin- and ALDH(hi)CD133+Lin- cells demonstrated distinct clonogenic progenitor function in vitro, whereas only the ALDH(hi)CD133+Lin- population seeded the murine bone marrow 48 hours after transplantation. Significant human cell repopulation was observed only in NOD/SCID and NOD/SCID beta2M-null mice that received transplants of ALDH(hi)CD133+Lin- cells. Limiting dilution analysis demonstrated a 10-fold increase in the frequency of NOD/SCID repopulating cells compared with CD133+Lin- cells, suggesting that high ALDH activity further purified cells with repopulating function. Transplanted ALDH(hi)CD133+Lin- cells also maintained primitive hematopoietic phenotypes (CD34+CD38-) and demonstrated enhanced repopulating function in recipients of serial, secondary transplants. Cell selection based on ALDH activity and CD133 expression provides a novel purification of HSCs with long-term repopulating function and may be considered an alternative to CD34 cell selection for stem cell therapies.

Purpose of Review: Exciting advances have been made in the field of hematopoietic stem cell biology during the past year. This review summarizes recent progress in the identification, culture, and in vivo tracking of hematopoietic stem cells., Recent Findings: The roles of Wnt and Notch proteins in regulating stem cell renewal in the microenvironment, and how these molecules can be exploited in ex vivo stem cell culture, are reviewed. The importance of identification of stem cells using functional as well as phenotypic markers is discussed. The novel field of nanotechnology is then discussed in the context of stem cell tracking in vivo. This review concludes with a section on the unexpected potential of bone marrow-derived stem cells to contribute to the repair of damaged tissues. The contribution of cell fusion to explain the latter phenomenon is discussed., Summary: Because of exciting discoveries made recently in the field of stem cell biology, researchers now have improved tools to define novel populations of stem cells, examine them ex vivo using conditions that promote self-renewal, track them into recipients, and determine whether they can contribute to the repair of damaged tissues. These discoveries will significantly advance the field of stem cell transplantation.

Human hematopoietic stem cells (HSCs) are commonly purified by the expression of cell surface markers such as CD34. Because cell phenotype can be altered by cell cycle progression or ex vivo culture, purification on the basis of conserved stem cell function may represent a more reliable way to isolate various stem cell populations. We have purified primitive HSCs from human umbilical cord blood (UCB) by lineage depletion (Lin(-)) followed by selection of cells with high aldehyde dehydrogenase (ALDH) activity. ALDH(hi)Lin(-) cells contained 22.6% +/- 3.0% of the Lin(-) population and highly coexpressed primitive HSC phenotypes (CD34(+) CD38(-) and CD34(+)CD133(+)). In vitro hematopoietic progenitor function was enriched in the ALDH(hi)Lin(-) population, compared with ALDH(lo)Lin(-) cells. Multilineage human hematopoietic repopulation was observed exclusively after transplantation of ALDH(hi)Lin(-) cells. Direct comparison of repopulation with use of the nonobese diabetic/severe combined immunodeficient (NOD/SCID) and NOD/SCID beta2 microglobulin (beta2M) null models demonstrated that 10-fold greater numbers of ALDH(hi)-Lin(-) cells were needed to engraft the NOD/SCID mouse as compared with the more permissive NOD/SCID beta2M null mouse, suggesting that the ALDH(hi)Lin(-) population contained committed progenitors as well as primitive repopulating cells. Cell fractionation according to lineage depletion and ALDH activity provides a viable and prospective purification of HSCs on the basis of cell function rather than cell surface phenotype.

The field of murine models of xenotransplantation has grown immensely over the past two decades. The explosive growth in this field is in part due to the fact that good in vitro methods do not exist yet to allow examination of human stem cell homing into the bone marrow compartment versus other tissues, long-term survival of human stem cells, or differentiation into tissues outside of the hematopoietic system. Since these important aspects of human stem cell biology can be examined in vivo using immune-deficient mice, the number of different strains and models is constantly increasing. The current review discusses the merits and drawbacks of each immune-deficient mouse xenograft system as it stands to date and reviews how each immune-deficient mouse model has been used to further our knowledge of human hematopoietic stem cell biology.

The cell surface protein CD34 is frequently used as a marker for positive selection of human hematopoietic stem/progenitor cells in research and in transplantation. However, populations of reconstituting human and murine stem cells that lack cell surface CD34 protein have been identified. In the current studies, we demonstrate that CD34 expression is reversible on human hematopoietic stem/progenitor cells. We identified and functionally characterized a population of human CD45(+)/CD34(-) cells that was recovered from the bone marrow of immunodeficient beige/nude/xid (bnx) mice 8 to 12 months after transplantation of highly purified human bone marrow-derived CD34(+)/CD38(-) stem/progenitor cells. The human CD45(+) cells were devoid of CD34 protein and mRNA when isolated from the mice. However, significantly higher numbers of human colony-forming units and long-term culture-initiating cells per engrafted human CD45(+) cell were recovered from the marrow of bnx mice than from the marrow of human stem cell-engrafted nonobese diabetic/severe combined immunodeficient mice, where 24% of the human graft maintained CD34 expression. In addition to their capacity for extensive in vitro generative capacity, the human CD45(+)/CD34(-) cells recovered from the bnx bone marrow were determined to have secondary reconstitution capacity and to produce CD34(+) progeny following retransplantation. These studies demonstrate that the human CD34(+) population can act as a reservoir for generation of CD34(-) cells. In the current studies we demonstrate that human CD34(+)/CD38(-) cells can generate CD45(+)/CD34(-) progeny in a long-term xenograft model and that those CD45(+)/CD34(-) cells can regenerate CD34(+) progeny following secondary transplantation. Therefore, expression of CD34 can be reversible on reconstituting human hematopoietic stem cells.

The immortalized murine stromal cell line AFT024 has been reported to maintain human hematopoietic progenitors in an undifferentiated state in vitro. In the current studies the beige/nude/xid (bnx) mouse in vivo xenograft model was used to examine the engraftment and multilineage generative potential of human hematopoietic progenitors after 2-3 weeks growth on AFT024 stroma, in comparison to primary stromal monolayers derived from post-natal human bone marrow. Eight to 12 months after transplantation of human CD34+CD38- cells from umbilical cord blood, cultured on AFT024 vs human stroma for 2-3 weeks, the murine bone marrow was harvested and analyzed for the presence of human myeloid and lymphoid cells. The mean percent engraftment of total human hematopoietic cells in the murine marrow was significantly higher after co-cultivation on AFT024 than on human stroma. Human myeloid and lymphoid lineage cells were detected in all mice. However, engraftment of myeloid lineage cells (CD33+), B lymphoid (CD19+), and T lymphoid cells (CD4+and CD8+) were significantly higher after co-cultivation of the human cells on AFT024 than on human stroma, prior to transplantation. Interestingly, the length of time in culture did not significantly affect the engraftment of the myeloid and T lymphoid lineage progenitors, but the percentage of B lymphoid lineage engraftment decreased significantly between 2 and 3 weeks of co-cultivation on both types of stroma. Cells with a primitive phenotype (CD45+/CD34-/CD38- and CD45+/CD34-/lin-) and cells with the capacity to generate secondary human CFU after recovery from the bnx bone marrow were maintained at significantly higher levels during culture on AFT024 stroma than on human stroma. The current studies demonstrate that the AFT024 murine stromal cell line supports the ex vivo survival and maintenance of human hematopoietic progenitors that are capable of long-term multilineage reconstitution for 2-3 weeks ex vivo, to levels superior to those that can be obtained using human stromal cells.

The mechanisms by which transforming growth factor beta (TGF-beta) exerts a negative effect on cell-cycle entry in primary human hematopoietic stem/progenitor cells were examined at the molecular and cellular levels. After treatment of primary human CD34+ progenitors with TGF-beta there was a decrease in the levels of cyclin D2 protein and an increase in levels of the cyclin-dependent kinase inhibitor (CDKI) p15 as compared to the levels in untreated cells. The converse was true after addition of neutralizing anti-TGF-beta antibody. Administration of TGF-beta to CD34+ cells in the presence of cytokines prevented retinoblastoma protein (pRb) phosphorylation, which occurred in the same cells treated with cytokines alone or cytokines and anti-TGF-beta antibody. Neutralization of TGF-beta during 24 to 48 hours of culture with cytokines significantly increased the number of colony-forming progenitors, but did not modulate the human stem cell pool, as measured in 6- to 12-month xenotransplantation assays. Equivalent numbers of human B, T, and myeloid cells were obtained after transplantation of cells treated with or without neutralization of TGF-beta.

Recent evidence suggests that expression of CD34 on the cell membrane does not always correlate with stem cell activity. In the mouse, there is a highly quiescent population of stem cells that lacks CD34 expression, but has full reconstituting capacity. The current review addresses the discovery of a similar population of dormant CD34-negative human hematopoietic stem cells. This information casts some uncertainty on the benefits of CD34+ cell isolation for stem cell transplantation, until more is known about the novel CD34-negative stem cell population. Methods designed to achieve removal of specific mature blood cell lineages might prove to be most advantageous in the future.

The lack of human B lymphocyte development in beige/nude/XID (bnx) mice is in sharp contrast to the robust development observed in another immune deficient strain, the NOD/SCID mouse. The ability to generate human B lymphocytes in the NOD/SCID, but not bnx mouse has been hypothesized to be caused by differences in the microenvironments or systemic cytokine concentrations. In the current studies we report that the differences in development can be primarily attributed to the source of the progenitors transplanted into the mice. The prior studies in bnx mice used cultured pediatric or adult bone marrow (BM) as the source of the CD34+ cells, whereas the NOD/SCID studies have predominantly used fresh or cultured umbilical cord blood (UCB). We have analyzed BM and UCB for the number of human CD34+/CD38- cells capable of in vitro B lymphocyte development, and have found a lower frequency of B lymphocyte generation in BM. The individual B lymphocyte clones that developed from bone marrow produced 100-fold fewer cells than the UCB-derived clones. In agreement with the in vitro studies, human B lymphocytes developed in bnx mice from both CD34+ and CD34+/CD38- cells isolated from human umbilical cord blood, but not from equivalent numbers of CD34+ and CD34+/CD38- progenitors from bone marrow. Therefore, the lower generative capacity, and frequency of B lymphocyte precursors in human marrow may be responsible for the previous results that showed a lack of B lymphocyte development in bnx mice.

Recent reports have indicated that there is poor engraftment from hematopoietic stem cells (HSC) that have traversed cell cycle ex vivo. However, inducing cells to cycle in culture is critical to the fields of ex vivo stem cell expansion and retroviral-mediated gene therapy. Through the use of a xenograft model, the current data shows that human hematopoietic stem and progenitor cells can traverse M phase ex vivo, integrate retroviral vectors, engraft, and sustain long-term hematopoiesis only if they have had the opportunity to engage their integrin receptors to fibronectin during the culture period. If cultured in suspension under the same conditions, transduction is undetectable and the long-term multilineage regenerative capacity of the primitive cells is severely diminished.

Successful gene therapy depends on stable transduction of hematopoietic stem cells. Target cells must cycle to allow integration of Moloney-based retroviral vectors, yet hematopoietic stem cells are quiescent. Cells can be held in quiescence by intracellular cyclin-dependent kinase inhibitors. The cyclin-dependent kinase inhibitor p15(INK4B) blocks association of cyclin-dependent kinase (CDK)4/cyclin D and p27(kip-1) blocks activity of CDK2/cyclin A and CDK2/cyclin E, complexes that are mandatory for cell-cycle progression. Antibody neutralization of beta transforming growth factor (TGFbeta) in serum-free medium decreased levels of p15(INK4B) and increased colony formation and retroviral-mediated transduction of primary human CD34(+) cells. Although TGFbeta neutralization increased colony formation from more primitive, noncycling hematopoietic progenitors, no increase in M-phase-dependent, retroviral-mediated transduction was observed. Transduction of the primitive cells was augmented by culture in the presence of antisense oligonucleotides to p27(kip-1) coupled with TGFbeta-neutralizing antibodies. The transduced cells engrafted immune-deficient mice with no alteration in human hematopoietic lineage development. We conclude that neutralization of TGFbeta, plus reduction in levels of the cyclin-dependent kinase inhibitor p27, allows transduction of primitive and quiescent hematopoietic progenitor populations.

The ultimate goal of human gene therapy in treating hematopoietic disorders is to insert a functional copy of the affected gene into self-renewing stem cells. The engineered pluripotent cells should then provide all lineages of corrected blood cells for the lifetime of the recipient. It is therefore important to develop methods of tracking and studying the progeny of individual human hematopoietic stem cells. Using the technique of single-colony inverse polymerase chain reaction (PCR), we assessed the clonal diversity of marked colony-forming cells that had developed from transduced human hematopoietic progenitors in a long-term xenograft system. The LN retroviral vector, which carries the neo gene, was used to individually mark human CD34+ progenitors. The marked cells were then transplanted into immune-deficient mice for periods of up to 1 year to assess their survival and retention of clonogenic capacity. Following long-term engraftment, bone marrow cells recovered from each mouse were plated in human-specific colony-forming unit (CFU) assay with and without the drug G418, which selects for cells expressing the neo gene. Three weeks later, well-isolated colonies that had grown in G418 were plucked, and PCR for the neo gene was performed to confirm the presence of the vector. Inverse PCR was then performed on neo+ colonies to analyze the integration site of the LN provirus in human DNA. The clonal diversity of G418-resistant (G418R) human CFU recovered from 18 long-term engrafted beige/nude/xid (bnx) mice was assessed. From one to six human hematopoietic precursors had generated all marked colony-forming progenitors (3-39) recovered from the marrow of each animal. To assess the extent of in vitro self-renewal divisions, marrow samples from 22 sets of experiments, with 2-4 mice transplanted in each set, were studied using the single-colony inverse PCR technique. Proviral integrants at identical sites were found in only two mice transplanted with cells transduced in the same flask. The presence of identical integration sites in human progenitors recovered from two mice demonstrated that a long-lived, marked cell had self-renewed in vitro before transplantation and that both daughter cells had retained the capacity to home to the bnx bone marrow and survive for 10 months. Our in vivo xenograft model and the inverse PCR technique have allowed us to identify, trace, and quantitate the clonogenic progeny of primitive human hematopoietic cells for up to 1 year after retroviral-mediated transduction.

Interleukin 3 (IL-3) supports the survival of multilineage hematopoietic progenitors, and increases the extent of retrovirally mediated gene transfer into colony-forming cells. However, effects from the supraphysiological levels used in ex-vivo expansion and gene-therapy protocols on subsequent differentiation of human progenitors have not been well defined. In the current studies, the extents of retrovirally mediated transduction and lineage development from CD34+ cells cultured ex vivo in the presence or absence of IL-3 were compared. All transductions were performed in the presence of an irradiated stromal support layer, IL-6 and SCF, with and without inclusion of 10 ng/ml IL-3. Following transduction, colony-forming (CFU) assays were done, and the remaining cells were transplanted into cohorts of sibling beige/nude/xid (bnx) mice. Marrow from the mice was harvested 9-10 months post transplantation. The average extent of human CD45+ cell engraftment in the bone marrow and the human hematopoietic lineages recovered from the mice in the +IL-3 and -IL-3 groups did not vary significantly. No deleterious effects on the extent of engraftment, lineage generation, or survival of clonogenic progenitors was observed with inclusion of IL-3 in the transductions performed on stromal support. The percentage of G418-resistant human progenitors recovered from mice was equivalent. The extent of marking by the neo gene in the marrow of the mice was equal in both groups, and inverse PCR revealed that primitive cells transduced in the absence of IL-3 had generated progeny with slightly better clonal diversity than progenitors transduced in the presence of IL-3. These data show that, while transduction of colony-forming progenitors may not always be apparent, primitive human hematopoietic cells can be transduced to significant levels in the absence of IL-3.

Stromal support is required during retroviral-mediated transduction of human bone marrow-derived CD34+ cells to maintain the clonogenicity of the primitive progenitors. We hypothesized that the cytokine FLT3 ligand (FL) might be able to replace the maintenance role provided by the stroma. CD34+ progenitors from human bone marrow were transduced by the retroviral vector LN with the cytokines interleukin-3 (IL-3), IL-6, and stem cell factor (SCF) present in all cultures. Transductions were performed with or without stromal support and with or without the inclusion of 100 U/mL FL. No significant increase in gene transfer into colony-forming cells was obtained by the addition of FL to the cultures. Transduction and survival of more primitive human hematopoietic cells was determined by growth in immune-deficient mice for 7 to 8 months. Human myeloid cells, T lymphocytes, and colony-forming progenitors were recovered from the marrow of mice that had received human cells transduced on stroma or in suspension culture with IL-3, IL-6, SCF, and FL, but not with IL-3, IL-6, and SCF alone. LN provirus was detected by polymerase chain reaction in the marrow recovered from 9 of 10 mice transplanted with human CD34+ cells transduced with stromal support, 5 of 11 mice that received human cells transduced in suspension culture with FL, but none of the 10 mice that received human cells transduced in suspension culture without FL We conclude that FLT3 ligand, in conjunction with IL-3, IL-6, and SCF, preserves the generative capacity of primitive human hematopoietic cells during in vitro transductions in suspension culture.

Gene transduction of pluripotent human hematopoietic stem cells (HSCs) is necessary for successful gene therapy of genetic disorders involving hematolymphoid cells. Evidence for transduction of pluripotent HSCs can be deduced from the demonstration of a retroviral vector integrated into the same cellular chromosomal DNA site in myeloid and lymphoid cells descended from a common HSC precursor. CD34+ progenitors from human bone marrow and mobilized peripheral blood were transduced by retroviral vectors and used for long-term engraftment in immune-deficient (beige/nude/XIS) mice. Human lymphoid and myeloid populations were recovered from the marrow of the mice after 7-11 months, and individual human granulocyte-macrophage and T-cell clones were isolated and expanded ex vivo. Inverse PCR from the retroviral long terminal repeat into the flanking genomic DNA was performed on each sorted cell population. The recovered cellular DNA segments that flanked proviral integrants were sequenced to confirm identity. Three mice were found (of 24 informative mice) to contain human lymphoid and myeloid populations with identical proviral integration sites, confirming that pluripotent human HSCs had been transduced.

We sought to define optimal conditions for retroviral-mediated transduction of long-lived human hematopoietic progenitors from bone marrow and peripheral blood. CD34+ cells were transduced by the LN and G2 retroviral vectors in the presence or absence of stromal support and with or without cytokine addition. After transduction, a portion of the cells was plated in methylcellulose colony-forming assay, with or without G418, to assess the extent of gene transfer into committed progenitors. The remaining cells from each experiment were transplanted into immunodeficient mice to allow analysis of transduction of long-lived progenitors. Human colony-forming cells contained within the murine bone marrow were analyzed after engraftment periods of 2 to 11 months. Cells were plated in a human-specific colony-forming assay with and without G418 to assess the extent of transduction of primitive progenitors. Individual human colonies were also analyzed by polymerase chain reaction for the presence of provirus. Bone marrow progenitors were efficiently transduced only when stroma was present, whereas mobilized peripheral blood progenitors were effectively transduced in the presence of either stroma or cytokines. Inclusion of the cytokines interleukin-3, interleukin-6, and stem cell factor did not further augment the extent of gene transfer in the presence of a stromal support layer. Additionally, human CD34+ progenitors from bone marrow or mobilized peripheral blood that had been transduced for 3 days in the absence of stroma failed to produce sustained, long-term engraftment of bnx mice. Mice transplanted with the same pools of human progenitors that had been transduced in the presence of stroma for 3 days had significant levels of human cell engraftment at the same timepoints, 7 to 11 months after transplantation. Our data show loss of long-lived human progenitors during 3-day in vitro transduction periods in the absence of stromal support. Therefore, the presence of bone marrow stroma has dual benefits in that it increases gene transfer efficiency and is essential for survival of long-lived human hematopoietic progenitors.

Human umbilical cord blood (UCB) can be a source of hematopoietic stem cells for gene therapy, as an alternative to allogeneic bone marrow transplantation, for the treatment of a number of genetic diseases. To determine conditions that yield maximal gene transfer into UCB progenitor cells, we examined a number of variables. We used cell-free retroviral vector supernatants that convey neomycin (G418) resistance and measured the percentage of G418-resistant progenitor-derived colonies. Adding retroviral supernatant to the UCB cells in basal medium once a day for 3 days produced a threefold increase of G418-resistant colonies (9.8%) compared to a single exposure to supernatant (3.1%). To establish whether recombinant human growth factors are beneficial during transduction, the presence of interleukin-3 (IL-3), IL-6, and mast cell growth factor (MGF, a c-kit ligand) were compared in different combinations. Inclusion of the three factors together caused a threefold increase of gene transfer (30.4%) compared to transduction in basal medium. When the UCB cells were precultured in medium containing IL-3, IL-6, and MGF for 3 days before addition of the retroviral supernatant on days 4, 5, and 6, the average extent of gene transfer was 21.8%, compared with an average of 34.4% when UCB cells were transduced on days 1, 2, and 3. The presence of marrow stroma during the transduction of the UCB cells did not further increase gene transfer. We conclude that UCB progenitor cells can be efficiently transduced with the use of recombinant human growth factors IL-3, IL-6, and MGF and may be a suitable source for gene therapy.

Retroviral vector-mediated gene transfer into human hematopoietic stem cells may permit gene therapy of numerous genetic diseases. Stimulation of marrow with hematopoietic growth factors (HGFs) has been shown to increase the level of retroviral transduction. We have examined the effects of recombinant human mast cell growth factor (MGF), alone and in combination with other HGFs, on the efficiency of gene transfer into human hematopoietic progenitor cells. MGF acts in concert with interleukin 3 (IL-3) and interleukin 6 (IL-6) to increase the percentage of CD34+ progenitors transduced with a retroviral vector expressing the neo gene. The most potent combination of growth factors that we examined, interleukin 1 (IL-1)/IL-3/IL-6/MGF, resulted in the conferral of G418 resistance to 45% of progenitors and long-term culture-initiating cells. Extending the time of cocultivation of the marrow cells with the vector-producing cells did not further increase gene transfer frequency, suggesting that the amount of available vector is not limiting. To analyze the effects of the HGF on gene transfer into more primitive hematopoietic progenitors, CD34+ cells were isolated from marrow samples that were purged of committed progenitor cells by treatment with 4-hydroperoxycyclophosphamide (4-HC). Preculturing the CD34+ 4-HC-treated cells with the combination of four HGF (IL-1/IL-3/IL-6/MGF) permitted transduction of 20%-28% of the progenitors that formed colonies after 30 days in culture. These results demonstrate that MGF in combination with other HGFs enhances gene transduction of human hematopoietic progenitor cells.

The human glucocerebrosidase (GC) gene has been expressed in the progeny of murine hematopoietic stem cells following transduction of marrow with a retroviral vector (G2) containing the human GC cDNA. Murine marrow was transduced via co-cultivation following prestimulation in the presence or absence of recombinant IL-3 and IL-6. A high rate of gene transfer and expression (95%) was demonstrated in primary day 12 CFU-S foci following bone marrow transplantation (BMT) of G2-transduced marrow into lethally irradiated syngeneic recipient mice. Immunoreactive human GC protein was also documented in the CFU-S foci. Primary recipient mice were examined 4-6 months following BMT. A higher rate of gene transfer (87%) was seen in hematopoietic organs of recipients of prestimulated donor marrow compared with organs from initially unstimulated marrow (25%). A high rate of expression of human GC was also documented in the prestimulated organs (50%) when compared with the unstimulated group (25%). Secondary BMT was performed using marrow from the long-lived primary recipients. The human GC gene was present in 88% of secondary day 12 CFU-S foci examined in the prestimulated group versus 23% in the unstimulated group. Expression of the human GC gene was documented in secondary day 12 CFU-S foci, providing strong evidence of initial hematopoietic stem cell transduction.

We have examined the ability of the recombinant hematopoietic growth factors (HGF) interleukin-3 (IL-3), IL-6, and granulocyte-macrophage colony-stimulating factor (GM-CSF) to increase retroviral vector-mediated gene transfer into human hematopoietic progenitor cells (HPC). The efficiency of neo gene transfer by the N2 vector into human HPC was enhanced by preculture with either GM-CSF or IL-3 (but not IL-6) and with each combination of the three factors. The combination of IL-3 plus IL-6 consistently produced significantly higher levels of G418-resistant colonies (50-60%) than any of the other combinations of HGF tested. Following preculture with HGF and transduction by N2, marrow was maintained in long-term bone marrow culture (LTBMC) for 2 months. The levels of G418-resistant HPC remained stable, and no apparent depletion of total HPC content resulted from the prior exposure to highly stimulatory doses of factors. The proliferative status of the HPC, following exposure to the HGF, was measured as the percentage of HPC that were inhibited from forming colonies by exposure to the S-phase-specific drug, hydroxyurea. The ability of the different HGF to increase the rate of gene transfer by N2 correlated significantly with the extent to which they stimulated HPC proliferation. These results suggest that the mechanism by which HGF increase rates of gene transfer into HPC is by stimulating cell proliferation. Techniques that produce high rates of gene transfer into long-lived human HPC will facilitate studies to quantitate expression of exogenous genes in hematopoietic cells and may be applicable to clinical gene therapy.