Preselection of retrovirally transduced bone marrow avoids subsequent stem cell gene silencing and age-dependent extinction of expression of human β-globin in engrafted mice

Transcriptional silencing of genes transferred into hematopoietic stem cells poses one of the most significant challenges to the success of gene therapy. If the transferred gene is not completely silenced, a progressive decline in gene expression as the mice age often is encountered. These phenomena were observed to various degrees in mouse transplant experiments using retroviral vectors containing a human β-globin gene, even when cis-linked to locus control region derivatives. Here, we have investigated whether ex vivo preselection of retrovirally transduced stem cells on the basis of expression of the green fluorescent protein driven by the CpG island phosphoglycerate kinase promoter can ensure subsequent long-term expression of a cis-linked β-globin gene in the erythroid lineage of transplanted mice. We observed that 100% of mice (n = 7) engrafted with preselected cells concurrently expressed human β-globin and the green fluorescent protein in 20–95% of their RBC for up to 9.5 mo posttransplantation, the longest time point assessed. This expression pattern was successfully transferred to secondary transplant recipients. In the presence of β-locus control region hypersensitive site 2 alone, human β-globin mRNA expression levels ranged from 0.15% to 20% with human β-globin chains detected by HPLC. Neither the proportion of positive blood cells nor the average expression levels declined with time in transplanted recipients. Although suboptimal expression levels and heterocellular position effects persisted, in vivo stem cell gene silencing and age-dependent extinction of expression were avoided. These findings support the further investigation of this type of vector for the gene therapy of human hemoglobinopathies.

Regulated expression of P210 Bcr-Abl during embryonic stem cell differentiation stimulates multipotential progenitor expansion and myeloid cell fate

P210 Bcr-Abl is an activated tyrosine kinase oncogene encoded by the Philadelphia chromosome associated with human chronic myelogenous leukemia (CML). The disease represents a clonal disorder arising in the pluripotent hematopoietic stem cell. During the chronic phase, patients present with a dramatic expansion of myeloid cells and a mild anemia. Retroviral gene transfer and transgenic expression in rodents have demonstrated the ability of Bcr-Abl to induce various types of leukemia. However, study of human CML or rodent models has not determined the direct and immediate effects of Bcr-Abl on hematopoietic cells from those requiring secondary genetic or epigenetic changes selected during the pathogenic process. We utilized tetracycline-regulated expression of Bcr-Abl from a promoter engineered for robust expression in primitive stem cells through multilineage blood cell development in combination with the in vitro differentiation of embryonal stem cells into hematopoietic elements. Our results demonstrate that Bcr-Abl expression alone is sufficient to increase the number of multipotent and myeloid lineage committed progenitors in a dose-dependent manner while suppressing the development of committed erythroid progenitors. These effects are reversible upon extinguishing Bcr-Abl expression. These findings are consistent with Bcr-Abl being the sole genetic change needed for the establishment of the chronic phase of CML and provide a powerful system for the analysis of any genetic change that alters cell growth and lineage choices of the hematopoietic stem cell.

Spermatogonial stem cell enrichment by multiparameter selection of mouse testis cells

The spermatogonial stem cell initiates and maintains spermatogenesis in the testis. To perform this role, the stem cell must self replicate as well as produce daughter cells that can expand and differentiate to form spermatozoa. Despite the central importance of the spermatogonial stem cell to male reproduction, little is known about its morphological or biochemical characteristics. This results, in part, from the fact that spermatogonial stem cells are an extremely rare cell population in the testis, and techniques for their enrichment are just beginning to be established. In this investigation, we used a multiparameter selection strategy, combining the in vivo cryptorchid testis model with in vitro fluorescence-activated cell sorting analysis. Cryptorchid testis cells were fractionated by fluorescence-activated cell sorting analysis based on light-scattering properties and expression of the cell surface molecules α6-integrin, αv-integrin, and the c-kit receptor. Two important observations emerged from these analyses. First, spermatogonial stem cells from the adult cryptorchid testis express little or no c-kit. Second, the most effective enrichment strategy, in this study, selected cells with low side scatter light-scattering properties, positive staining for α6-integrin, and negative or low αv-integrin expression, and resulted in a 166-fold enrichment of spermatogonial stem cells. Identification of these characteristics will allow further purification of these valuable cells and facilitate the investigation of molecular mechanisms governing spermatogonial stem cell self renewal and hierarchical differentiation.

N-terminal transcriptional activation domain of LZIP comprises two LxxLL motifs and the Host Cell Factor-1 binding motif

Host Cell Factor-1 (HCF-1, C1) was first identified as a cellular target for the herpes simplex virus transcriptional activator VP16. Association between HCF and VP16 leads to the assembly of a multiprotein enhancer complex that stimulates viral immediate-early gene transcription. HCF-1 is expressed in all cells and is required for progression through G1 phase of the cell cycle. In addition to VP16, HCF-1 associates with a cellular bZIP protein known as LZIP (or Luman). Both LZIP and VP16 contain a four-amino acid HCF-binding motif, recognized by the N-terminal β-propeller domain of HCF-1. Herein, we show that the N-terminal 92 amino acids of LZIP contain a potent transcriptional activation domain composed of three elements: the HCF-binding motif and two LxxLL motifs. LxxLL motifs are found in a number of transcriptional coactivators and mediate protein–protein interactions, notably recognition of the nuclear hormone receptors. LZIP is an example of a sequence-specific DNA-binding protein that uses LxxLL motifs within its activation domain to stimulate transcription. The LxxLL motifs are not required for association with the HCF-1 β-propeller and instead interact with other regions in HCF-1 or recruit additional cofactors.

Rac and Cdc42 GTPases control hematopoietic stem cell shape, adhesion, migration, and mobilization

Critical to homeostasis of blood cell production by hematopoietic stem/progenitor (HSC/P) cells is the regulation of HSC/P retention within the bone marrow microenvironment and migration between the bone marrow and the blood. Key extracellular regulatory elements for this process have been defined (cell–cell adhesion, growth factors, chemokines), but the mechanism by which HSC/P cells reconcile multiple external signals has not been elucidated. Rac and related small GTPases are candidates for this role and were studied in HSC/P deficient in Rac2, a hematopoietic cell-specific family member. Rac2 appears to be critical for HSC/P adhesion both in vitro and in vivo, whereas a compensatory increase in Cdc42 activation regulates HSC/P migration. This genetic analysis provides physiological evidence of cross-talk between GTPase proteins and suggests that a balance of these two GTPases controls HSC/P adhesion and mobilization in vivo.

Remodeling of the postnatal mouse testis is accompanied by dramatic changes in stem cell number and niche accessibility

Little is known about stem cell biology or the specialized environments or niches believed to control stem cell renewal and differentiation in self-renewing tissues of the body. Functional assays for stem cells are available only for hematopoiesis and spermatogenesis, and the microenvironment, or niche, for hematopoiesis is relatively inaccessible, making it difficult to analyze donor stem cell colonization events in recipients. In contrast, the recently developed spermatogonial stem cell assay system allows quantitation of individual colonization events, facilitating studies of stem cells and their associated microenvironment. By using this assay system, we found a 39-fold increase in male germ-line stem cells during development from birth to adult in the mouse. However, colony size or area of spermatogenesis generated by neonate and adult stem cells, 2–3 months after transplantation into adult tubules, was similar (∼0.5 mm2). In contrast, the microenvironment in the immature pup testis was 9.4 times better than adult testis in allowing colonization events, and the area colonized per donor stem cell, whether from adult or pup, was about 4.0 times larger in recipient pups than adults. These factors facilitated the restoration of fertility by donor stem cells transplanted to infertile pups. Thus, our results demonstrate that stem cells and their niches undergo dramatic changes in the postnatal testis, and the microenvironment of the pup testis provides a more hospitable environment for transplantation of male germ-line stem cells.

Regulation of the stem cell leukemia (SCL) gene: A tale of two fishes

The stem cell leukemia (SCL) gene encodes a tissue-specific basic helix–loop–helix (bHLH) protein with a pivotal role in hemopoiesis and vasculogenesis. Several enhancers have been identified within the murine SCL locus that direct reporter gene expression to subdomains of the normal SCL expression pattern, and long-range sequence comparisons of the human and murine SCL loci have identified additional candidate enhancers. To facilitate the characterization of regulatory elements, we have sequenced and analyzed 33 kb of the SCL genomic locus from the pufferfish Fugu rubripes, a species with a highly compact genome. Although the pattern of SCL expression is highly conserved from mammals to teleost fish, the genes flanking pufferfish SCL were unrelated to those known to flank both avian and mammalian SCL genes. These data suggest that SCL regulatory elements are confined to the region between the upstream and downstream flanking genes, a region of 65 kb in human and 8.5 kb in pufferfish. Consistent with this hypothesis, the entire 33-kb pufferfish SCL locus directed appropriate expression to hemopoietic and neural tissue in transgenic zebrafish embryos, as did a 10.4-kb fragment containing the SCL gene and extending to the 5′ and 3′ flanking genes. These results demonstrate the power of combining the compact genome of the pufferfish with the advantages that zebrafish provide for studies of gene regulation during development. Furthermore, the pufferfish SCL locus provides a powerful tool for the manipulation of hemopoiesis and vasculogenesis in vivo.

Interleukin 3 or interleukin 1 abrogates the reconstituting ability of hematopoietic stem cells.

Because of their known myelopoietic activities, both interleukin (IL)-3 and IL-1 are often used in combination with other cytokines for in vitro (ex vivo) expansion of stem cells. We have investigated the effects of IL-3 and IL-1 on in vitro expansion of murine hematopoietic stem cells with long-term engraftment capabilities, using a highly purified progenitor population. Lineage-negative, Ly-6A/E+, c-kit+ bone marrow cells from male mice were cultured in suspension in the presence of stem cell factor, IL-6, IL-11, and erythropoietin with or without IL-3 or IL-1. Kinetic studies revealed an exponential increase in total nucleated cells and about 10-fold enhancement of nucleated cells by IL-3 during the initial 10 days. Addition of IL-3 hastened the development but significantly suppressed the peak production of colony-forming cells. Addition of IL-1 also significantly suppressed the numbers of colony-forming cells. The reconstituting ability of the cultured cells was tested by transplanting the expanded male cells into lethally irradiated female mice. The cells expanded from enriched cells in the absence of IL-3 and IL-1 revealed engraftment at 2, 4, 5, and 6 months, whereas addition of IL-3 or IL-1 to the cultures significantly reduced the reconstituting ability. The results suggest that these cytokines may have a modulatory role on the self-renewal of stem cells and further indicate that the use of IL-3 and IL-1 for in vitro expansion of human stem cells needs to be cautiously evaluated.

Apoptosis of hemopoietic cells by the human granulocyte-macrophage colony-stimulating factor mutant E21R.

Human granulocyte-macrophage colony-stimulating factor (GM-CSF) binds to a high-affinity heterodimeric receptor composed of a specific alpha chain and a common beta chain (beta(c)), which is shared with the receptors for interleukins 3 and 5. Hemopoietic cell survival requires GM-CSF binding this high-affinity receptor. We have recently developed the GM-CSF mutant E21R, which selectively binds to the alpha chain and behaves as a competitive GM-CSF antagonist. We have now examined the role of E21R on the survival of hemopoietic cells and found that E21R causes apoptosis (programmed cell death) of normal and malignant cells directly in the absence of GM-CSF. The direct apoptotic effect of E21R occurred in a dose- and time-dependent manner. Apoptosis by E21R was dependent on cells expressing the high-affinity GM-CSF receptor and could be blocked by GM-CSF. Significantly, apoptosis of the cells occurred even in the presence of the survival factors granulocyte CSF and stem cell factor but was prevented by engagement of beta(c) with interleukin 3. The initiation of apoptosis required phosphorylation, transcriptional activity, and protein synthesis. These findings support a model whereby binding of E21R to the alpha chain leads to apoptosis, while beta(c) plays an important role in cell survival. This model may be applicable to other multimeric cytokine receptors and offers a novel approach for the treatment of human leukemia.

Increasing DNA repair methyltransferase levels via bone marrow stem cell transduction rescues mice from the toxic effects of 1,3-bis(2-chloroethyl)-1-nitrosourea, a chemotherapeutic alkylating agent.

The chloroethylnitrosourea (CNU) alkylating agents are commonly used for cancer chemotherapy, but their usefulness is limited by severe bone marrow toxicity that causes the cumulative depletion of all hematopoietic lineages (pancytopenia). Bone marrow CNU sensitivity is probably due to the inefficient repair of CNU-induced DNA damage; relative to other tissues, bone marrow cells express extremely low levels of the O6-methylguanine DNA methyltransferase (MGMT) protein that repairs cytotoxic O6-chloroethylguanine DNA lesions. Using a simplified recombinant retroviral vector expressing the human MGMT gene under control of the phosphoglycerate kinase promoter (PGK-MGMT) we increased the capacity of murine bone marrow-derived cells to repair CNU-induced DNA damage. Stable reconstitution of mouse bone marrow with genetically modified, MGMT-expressing hematopoietic stem cells conferred considerable resistance to the cytotoxic effects of 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), a CNU commonly used for chemotherapy. Bone marrow harvested from mice transplanted with PGK-MGMT-transduced cells showed extensive in vitro BCNU resistance. Moreover, MGMT expression in mouse bone marrow conferred in vivo resistance to BCNU-induced pancytopenia and significantly reduced BCNU-induced mortality due to bone marrow hypoplasia. These data demonstrate that increased DNA alkylation repair in primitive hematopoietic stem cells confers multilineage protection from the myelosuppressive effects of BCNU and suggest a possible approach to protecting cancer patients from CNU chemotherapy-related toxicity.

The stem cell antigen CD34 functions as a regulator of hemopoietic cell adhesion.

Although the CD34 antigen is widely used in the identification and purification of hemopoietic stem and progenitor cells, its function within hemopoiesis is unknown. We have investigated this issue by ectopically expressing human (hu) CD34 on the surface of murine hemopoietic cells. Forced expression of hu-CD34 in the thymocytes of transgenic mice did not appear to affect the development, maturation, or distribution of murine T cells but did significantly increase their ability to adhere to bone marrow stromal layers of human but not mouse origin. Ectopic expression of hu-CD34 on murine 416B cells, a multipotential progenitor that expresses murine CD34, yielded similar results. In both cases hu-CD34-dependent adhesion was enhanced by molecular engagement of the hu-CD34 protein using anti-CD34 antibodies. These results provide evidence that CD34 promotes the adhesive interactions of hemopoietic cells with the stromal microenvironment of the bone marrow thereby implicating CD34 in regulation and compartmentalization of stem cells. We propose that CD34 regulates these processes in part via an indirect mechanism, signaling changes in cellular adhesion in response to molecular recognition of an as yet unidentified stromal CD34 counterreceptor or ligand.

Identification of a unique membrane-bound molecule on a hemopoietic stem cell line and on multipotent progenitor cells.

Hemopoietic stem cells are a distinct population of cells that can differentiate into multilineages of hemopoietic cells and have long-term repopulation capability. A few membrane-bound molecules have been found to be preferentially, but not uniquely, present on the surface of these primitive cells. We report here the identification of a unique 105-kDa glycoprotein on the surface of hemopoietic stem cell line BL3. This molecule, recognized by the absorbed antiserum, is not present on the surface of myeloid progenitors 32D and FDC-P1 cells, EL4 T cells, and NIH 3T3 fibroblasts. This antiserum can also be used to block the proliferation of BL3 cells even in the presence of mitogen-stimulated spleen cell conditioned medium, which is known to have a stimulating activity on BL3 cells. It can also inhibit development of in vitro, fetal liver cell-derived multilineage colonies, but not other types of colonies, and of in vivo bone marrow cell-derived colony-forming unit spleen foci. These data suggest that gp105 plays an important role in hemopoietic stem cell differentiation.

In vitro generation of hematopoietic stem cells from an embryonic stem cell line.

Hematopoietic stem cells (HSC) are unique in that they give rise both to new stem cells (self-renewal) and to all blood cell types. The cellular and molecular events responsible for the formation of HSC remain unknown mainly because no system exists to study it. Embryonic stem (ES) cells were induced to differentiate by coculture with the stromal cell line RP010 and the combination of interleukin (IL) 3, IL-6, and F (cell-free supernatants from cultures of the FLS4.1 fetal liver stromal cell line). Cell cytometry analysis of the mononuclear cells produced in the cultures was consistent with the presence of PgP-1+ Lin- early hematopoietic (B-220- Mac-1- JORO 75- TER 119-) cells and of fewer B-220+ IgM- B-cell progenitors and JORO 75+ T-lymphocyte progenitors. The cell-sorter-purified PgP-1+ Lin- cells produced by induced ES cells could repopulate the lymphoid, myeloid, and erythroid lineages of irradiated mice. The ES-derived PgP-1+ Lin- cells must possess extensive self-renewal potential, as they were able to produce hematopoietic repopulation of secondary mice recipients. Indeed, marrow cells from irradiated mice reconstituted (15-18 weeks before) with PgP-1+ Lin- cell-sorter-purified cells generated by induced ES cells repopulated the lymphoid, myeloid, and erythroid lineages of secondary mouse recipients assessed 16-20 weeks after their transfer into irradiated secondary mice. The results show that the culture conditions described here support differentiation of ES cells into hematopoietic cells with functional properties of HSC. It should now be possible to unravel the molecular events leading to the formation of HSC.

Long-term in vivo expression of the human glucocerebrosidase gene in nonhuman primates after CD34+ hematopoietic cell transduction with cell-free retroviral vector preparations.

Successful gene transfer into stem cells would provide a potentially useful therapeutic modality for treatment of inherited and acquired disorders affecting hematopoietic tissues. Coculture of primate bone marrow cells with retroviral producer cells, autologous stroma, or an engineered stromal cell line expressing human stem cell factor has resulted in a low efficiency of gene transfer as reflected by the presence of 0.1-5% of genetically modified cells in the blood of reconstituted animals. Our experiments in a nonhuman primate model were designed to explore various transduction protocols that did not involve coculture in an effort to define clinically useful conditions and to enhance transduction efficiency of repopulating cells. We report the presence of genetically modified cells at levels ranging from 0.1% (granulocytes) to 14% (B lymphocytes) more than 1 year following reconstitution of myeloablated animals with CD34+ immunoselected cells transduced in suspension culture with cytokines for 4 days with a retrovirus containing the glucocerebrosidase gene. A period of prestimulation for 7 days in the presence of autologous stroma separated from the CD34+ cells by a porous membrane did not appear to enhance transduction efficiency. Infusion of transduced CD34+ cells into animals without myeloablation resulted in only transient appearance of genetically modified cells in peripheral blood. Our results document that retroviral transduction of primate repopulating cells can be achieved without coculture with stroma or producer cells and that the proportion of genetically modified cells may be highest in the B-lymphoid lineage under the given transduction conditions.