A potential molecular approach to ex vivo hematopoietic expansion with recombinant epidermal growth factor receptor-expressing adenovirus vector

Ex vivo expansion of hematopoietic stem cell (HSC) is an attractive technology for its potency of a variety of clinical applications. Such a technology has been achieved to some extent with combinations of various cytokines or continuous perfusion cultures. However, much more improvement is required especially for expansion of primitive hematopoietic progenitors. We propose here a novel molecular approach that might have the potential to compensate the current expansion. We designed an adenovirus vector to transiently express human epidermal growth factor receptor (EGFR), which is known to transduce only a mitogenic, but not a differentiation signal to mouse bone marrow cells on human purified CD34+ peripheral blood (PB) cells, and tried to expand these cells with EGF ex vivo. Because we found that exposure of CD34+ PB cells to cytokines induced surface expression of adenovirus-internalization receptor and rendered these cells permissive to adenovirus infection, we infected these cells with the adenovirus vector carrying EGFR gene in the presence of cytokines. Two-color flow cytometric analysis demonstrated that 60.3% +/- 22.4% of CD34+ cells expressed the adenovirus-mediated EGFR. Moreover, long-term culture-initiating cell assay showed that adenovirus vector could transduce more primitive progenitors. Subsequently, we tried to expand these cells in suspension culture with EGF for 5 days. Methylcellulose clonal assay showed that EGF induced 5.0- +/- 2.4-fold proliferation of the colony-forming unit pool during 5 days of expansion. The simple procedure of efficient adenovirus gene delivery to immature hematopoietic cells proved promising, and this technique was potentially applicable for a novel strategy aiming at ex vivo expansion of hematopoietic progenitors.     

Comparison of the metabolism of retinol delivered to human keratinocytes either bound to serum retinol-binding protein or added directly to the culture medium

The potentiality of primitive human hematopoietic cells can be profoundly affected by in vitro culture. Due to the growing number of protocols proposed for stem cell gene therapy and ex vivo expansion, it is crucial to define methods to preserve the generative capacity of human stem cells in culture while promoting self-renewal divisions. Stem cell division, homing, and subsequent lineage development can only be studied definitively by marking of pluripotent cells, followed by tracking and clonal analysis of the progeny in a long-term transplantation system. We have developed a bnx/hu xenograft model, in which transduced human hematopoietic cells can be individually tracked into different lineages over the course of one year post-transplantation. The tracking is accomplished by single cell cloning of individual T lymphoid and myeloid progenitors recovered from the marrow of the mice, and clonal integration analysis by the sensitive technique of single-colony inverse PCR. All cells derived from a stem cell transduced by a retroviral vector will carry the unique restriction fragment length polymorphism (RFLP) created by the random integration event. We have used the bnx/hu xenograft system coupled with single-colony inverse PCR to determine that human stem cells require stromal support, fibronectin support with cytokines, or the presence of Flt3 ligand during a 72-h ex vivo culture to maintain the ability to sustain long-term multilineage hematopoiesis.     

Ex vivo culture of CD34+/Lin-/DR- cells in stroma-derived soluble factors, interleukin-3, and macrophage inflammatory protein-1alpha maintains not only myeloid but also lymphoid progenitors in a novel switch culture assay

We have demonstrated that long-term culture initiating cells (LTC-IC) are maintained in a stroma noncontact (SNC) culture where progenitors are separated from stroma by a microporous membrane and LTC-IC can proliferate if the culture is supplemented with interleukin-3 (IL-3) and macrophage inflammatory protein-1alpha (MIP-1alpha). We hypothesize that the same conditions, which result in LTC-IC proliferation, may also maintain lymphoid progenitors. Natural killer (NK) cells are of lymphoid lineage and a stromal-based culture can induce CD34+/Lin-/DR- cells to differentiate along the NK cell lineage. We developed a three-step switch culture assay that was required to demonstrate the persistence of NK progenitors in CD34+/Lin-/DR- cells assayed in SNC cultures supplemented with IL-3 and MIP-1alpha. When CD34+/Lin-/DR- progeny from the SNC culture were plated sequentially into "NK cell progenitor switch" conditions (contact with stromal ligands, hydrocortisone-containing long-term culture medium, IL-2, IL-7, and stem cell factor [SCF]) followed by "NK cell differentiation" conditions (contact with stromal ligands, human serum, no hydrocortisone, and IL-2), significant numbers of CD56+/CD3- NK resulted, which exhibited cytotoxic activity against K562 targets. All steps are required because a switch from SNC cultures with IL-3 and MIP-1alpha directly to "NK cell differentiation" conditions failed to yield NK cells suggesting that critical step(s) in lymphoid commitment were missing. Additional experiments showed that CD34+/CD33- cells present after SNC cultures with IL-3 and MIP-1alpha, which contained up to 30% LTC-IC, are capable of NK outgrowth using the three-step switch culture. Limiting dilution analysis from these experiments showed a cloning frequency within the cultured CD34+/CD33- population similar to fresh sorted CD34+/Lin-/DR- cells. However, after addition of FLT-3 ligand, the frequency of primitive progenitors able to develop along the NK lineage increased 10-fold. In conclusion, culture of primitive adult marrow progenitors ex vivo in stroma-derived soluble factors, MIP-1alpha, and IL-3 maintains both very primitive myeloid (LTC-IC) and lymphoid (NK) progenitors and suggests that these conditions may support expansion of human hematopoietic stem cells. Addition of FLT-3 ligand to IL-2, IL-7 SCF, and stromal factors are important in early stages of NK development.     

Modulation of plasma membrane H+-ATPase activity differentially activates wound and pathogen defense responses in tomato plants

Hematopoiesis is viewed as a differentiating system emanating from a pluripotent hematopoietic stem cell capable of both self-renewal and differentiation. By identifying and characterizing a novel and highly specific in vitro mitogenic response to the N-acetyl glucosamyl/sialic acid specific, stem cell-binding lectin wheat germ agglutinin (WGA), we demonstrate the existance of a rare (0.1%), plastic adherent precursor in rat bone marrow capable of proliferation (two to seven divisions) in response to WGA. Stimulated cells possess a lineage (lin)low/- immunophenotype and immature blastoid morphology (WGA blasts). A subsequent proliferative response to stem cell factor (SCF), the ligand for the proto-oncogene receptor tyrosine kinase c-kit, is characterized by an initial maturation in immunophenotype and subsequent self-renewal of cells (SCF blasts) without differentiation for at least 50 generations. Although granulocyte colony-stimulating factor (G-CSF), interleukin (IL) -6, IL-7, and IL-11 synergize with SCF to increase blast colony formation, cytokines such as granulocyte-macrophage CSF or IL-3 are without significant effect. At all time points in culture, however, cells rapidly differentiate to mature neutrophils with dexamethasone or to mainly monocytes/macrophages in the presence of 1alpha,25-dihydroxyvitamin D3, characterized by cell morphology and cytochemistry. Removal of SCF during blast maturation, self-renewal, or induction of differentiation phases results in apoptotic cell death. Data indicate a pivotal role for SCF/c-kit interaction during antigenic maturation, self-renewal, and apoptotic protection of these lineage-restricted progenitors during non-CSF-mediated induction of differentiation. This approach provides a source of many normal, proliferating myelomonocytic precursor cells, and introduces possible clinical applications of ex vivo expanded myeloid stem cells.     

The effects of Flk-2/flt3 ligand as compared with c-kit ligand on short-term and long-term proliferation of CD34+ hematopoietic progenitors elicited from human fetal liver, umbilical cord blood, bone marrow, and mobilized peripheral blood

The Flk-2/flt3 ligand (FL) was evaluated and compared with c-kit ligand (KL) for its in vitro proliferative effects on CD34+ cells from human fetal liver, umbilical cord blood, bone marrow, and mobilized peripheral blood. Using a 7-day liquid culture system, FL in combination with interleukin-3 (IL-3), interleukin-6 (IL-6), and granulocyte colony-stimulating factor (G-CSF) was comparable with KL in combination with IL-3, IL-6, and G-CSF for the expansion of hematopoietic progenitors. When FL-containing cultures were assayed after 21 or 28 days, a greater number of progenitors were generated as compared with KL-containing cultures. Using bone marrow microvascular endothelial cells as support stroma, cultures supplemented with FL generated a greater number of progenitors in both the nonadherent and adherent layers at day 35. These data suggest that FL ligand, in combination with other cytokines, can be used for short-term ex vivo expansion of hematopoietic progenitors and facilitates the preservation and possible expansion of primitive cells capable of long-term generation of progenitors.     

A tumor necrosis factor-responsive long-term-culture-initiating cell is associated with the stromal layer of mouse long-term bone marrow cultures

Long-term bone marrow cultures provide a model for the study of hematopoiesis. Both an intact, adherent stromal layer and hematopoietic stem cells are necessary components in these cultures. Mycophenolic acid treatment of mouse long-term bone marrow cultures depletes them of all assayable hematopoietic precursors. The residual stromal cells are functional and support hematopoiesis if new progenitor cells are supplied. We now show that these mycophenolic acid-treated stromal cell cultures contain cells capable of hematopoietic differentiation without the addition of new progenitors. When treated with tumor necrosis factor alpha (20-200 units/ml), the apparently pure stromal cultures undergo an intense burst of hematopoietic activity. After 4 days such cultures contain approximately 2 x 10(6) hematopoietic cells and, by 1 week, they are indistinguishable from control long-term cultures that were not treated with mycophenolic acid. These results suggest that the stromal cultures either contain hematopoietic stem cells that are maintained quiescent and mycophenolic acid-resistant, perhaps by intimate contact with the stroma, or contain adherent cells that can be induced to differentiate into hematopoietic stem cells. These stem cells are primitive, in that they are capable of multilineage development in the long-term cultures, but are unable to form spleen colonies or myeloid colonies in semisolid medium. These data demonstrate that the adherent fraction of cultured bone marrow contains very primitive hematopoietic cells and that tumor necrosis factor alpha activates their proliferation and differentiation. They also suggest a strategy for obtaining the earliest progenitors free of other, more mature cell types.     

Ability of early acting cytokines to directly promote survival and suppress apoptosis of human primitive CD34+CD38- bone marrow cells with multilineage potential at the single-cell level: key role of thrombopoietin

Purified primitive progenitor/stem cells from bone marrow represent likely target populations for ex vivo expansion of stem cells to be used in high-dose chemotherapy or gene therapy. Whereas such primitive progenitor cells require combined stimulation by multiple cytokines for growth, some cytokines selectively promote viability rather than growth when acting individually. We investigated here for the first time the direct effects of cytokines on survival of primitive CD34+CD38- human bone marrow progenitor cells at the single-cell level. Interleukin-3 (IL-3) and the ligands for c-kit (KL) and flt3 (FL) had direct and selective viability-promoting effects on a small fraction of CD34+CD38- but not CD34+CD38+ progenitor cells. Interestingly, the recently cloned thrombopoietin (Tpo), although stimulating little growth, kept most CD34+CD38- progenitors viable after prolonged culture, maintaining twofold and fourfold more progenitors viable than KL and IL-3, respectively. A high fraction of these progenitors had a combined myeloid and erythroid differentiation potential, as well as capacity for prolonged production of progenitor cells under stroma-independent conditions. In addition, Tpo promoted viability of CD34+CD38- long-term culture-initiating cells, further supporting the idea that Tpo promotes viability of primitive human progenitor cells. Finally, Tpo suppressed apoptosis of CD34+CD38- cells in culture. Thus, the present studies show a novel effect of Tpo, implicating a potential role of this cytokine in maintaining quiescent primitive human progenitor cells viable.     

Distinct actions of interleukin-9 and interleukin-4 on a hematopoietic stem cell line, EMLC1

EMLC1 is a hematopoietic stem cell line that depends on stem cell factor (SCF) for growth and generates lymphoid, erythroid and myeloid progenitors in the presence of different cytokines. We have studied signaling events leading to cell proliferation and differentiation of EMLC1 mediated by interleukin (IL)-4 and IL-9. It was found that IL-9 enhances SCF-induced cell proliferation and promotes erythropoietin (EPO)-dependent erythroid differentiation of EMLC1 cells. However, IL-9 alone cannot support the growth of this cell line. In contrast, IL-4 by itself is sufficient to promote the growth of EMLC1 cells, even in the absence of SCF. Antiphosphotyrosine immunoblots of total cell lysates demonstrated that IL-4 and IL-9 induce tyrosine phosphorylation of different cellular substrates. Both IL-4 and IL-9 stimulated tyrosine phosphorylation of SHP-2, whereas the 90-kD tyrosine phosphorylated protein induced by IL-9 stimulation is Stat3. We have also shown that IL-4 is much more potent than IL-9 in inducing the expression of primary response gene c-myc. It was further determined that c-myc antisense oligodeoxynucleotide blocked IL-4 supported cell growth. Taken together, these results indicate that IL-4 may serve as a growth-promoting factor for hematopoietic stem cells, and IL-9 enhances both growth and erythroid differentiation of primitive hematopoietic progenitors. The results also suggest that differences in tyrosine phosphorylation induced by IL-4 and IL-9 may in part determine their distinct biological functions.     

Ex vivo expansion with stem cell factor and interleukin-11 augments both short-term recovery posttransplant and the ability to serially transplant marrow

The characterization of many cytokines involved in the control of hematopoiesis has led to intense investigation into their potential use in ex vivo culture to expand progenitor numbers. We have established the optimum ex vivo culture conditions that allow substantial amplification of transient engrafting murine stem cells and which, simultaneously, augment the ability to sustain serial bone marrow transplantation (BMT). Short-term incubation of unfractionated BM cells in liquid culture with stem cell factor (SCF) and interleukin-11 (IL-11) produced a 50-fold amplification of clonogenic multipotential progenitors (CFU-A). Following such ex vivo expansion, substantially fewer cells were required to rescue lethally irradiated mice. When transplanted in cell doses above threshold for engraftment, BM cells expanded ex vivo resulted in significantly more rapid hematopoietic recovery. In a serial transplantation model, unmanipulated BM was only able to consistently sustain secondary BMT recipients, but BM expanded ex vivo has sustained quaternary BMT recipients that remain alive and well more than 140 days after 4th degree BMT. These results show augmentation of both short-term recovery posttransplant and the ability to serially transplant marrow by preincubation in culture with SCF and IL-11.     

Adhesion to fibronectin maintains regenerative capacity during ex vivo culture and transduction of human hematopoietic stem and progenitor cells

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.     

Postnatal somatic growth and insulin contents in moderate or severe intrauterine growth retardation in the rat

Hematopoiesis in the vertebrate is characterized by the induction of ventral mesoderm to form hematopoietic stem cells and the eventual differentiation of these progenitors to form the peripheral blood lineages. Several genes have been implicated in the differentiation and development of hematopoietic and vascular progenitor cells, yet our understanding of the discrete steps involved in the induction of these cells from the ventral mesoderm is still incomplete. One method of delineating these processes is based on the use of lower vertebrates. The zebrafish (Danio rerio) is an especially robust vertebrate system for both isolating and characterizing genes involved in these processes. Hematopoietic mutants have been generated with defects in many of the steps of both the primitive and definitive hematopoietic programs. Cloning of the genes that underlie these mutations should yield valuable details of hematopoiesis and may have therapeutic implications for bone marrow transplantation and stem cell gene therapy.     

Thrombopoietin, steel factor and the ligand for flt3/flk2 interact to stimulate the proliferation of human hematopoietic progenitors in culture

We have tested the effects of steel factor (SF) the ligand for flt3/flk2 (FL) and thrombopoietin (TPO, Mpl ligand), on the proliferation of primitive human bone marrow progenitors in serum-deprived culture. Varying combinations of SF, FL and TPO supported formation of only few colonies from CD34+/c-Kit(low)/CD38neg/low cells. However, the addition of interleukin 3 (IL-3) to the three cytokines significantly increased the number of colonies. When this population of cells was tested in suspension culture for one week for production of colony-forming cells there was synergism among SF, FL and TPO. Addition of IL-3 to the three cytokines further increased the number of erythroid colony-forming cells. The effects of these four factors on CD34+/c-Kit(low)/CD38high cells were merely additive. Studies of individual CD34+/c-Kit(low)/CD38neg/low cells demonstrated the direct effects of SF, FL and TPO. In the presence of SF, FL and TPO, approximately half of the individual CD34+/c-Kit(low)/CD38neg/low cells proliferated in seven day suspension culture. Addition of IL-3 to the combination of SF, FL and TPO did not increase the frequencies of proliferating clones, but increased the size of individual clones. These observations suggest that SF, FL and TPO play important roles in survival and proliferation of primitive human hematopoietic progenitors.     

Giant cell fibroblastoma associated with dermatofibrosarcoma protuberans: a case report

Umbilical cord blood (UCB) was disclosed to possess the proliferative capacity containing hematopoietic progenitors and has recently been applied for allogeneic transplantation as an attractive alternative to bone marrow or peripheral blood stem cells. UCB contains similar and higher proportions of immature hematopoietic progenitors, compared to bone marrow stem cells, although the number of collectable cells is limited. The yield of collectable UCB volume ranges from 70 to 150 ml. The colony formation of CFU-Mix of UCB was higher, but that of CFU-GM and CFU-E was lower, compared to those of bone marrow. The analyses of expression of differentiation antigens and adhesion molecules on CD34+ cells of UCB by flow cytometer, revealed that the coexpression rates of CD38 and CD44 on CD34+ cells were almost the same, but the mean fluorescence intensity of those was low compared to adult bone marrow. These results indicate that UCB contains more primitive hematopoietic progenitors. UCB transplantation has greater advantages of lower incidences of graft versus host disease, and unlimited number of donors compared with other allogeneic transplantation would widen the indication of transplantation by technical and methodological development.     

Structurally specific heparan sulfates support primitive human hematopoiesis by formation of a multimolecular stem cell niche

Stem cell localization, conservation, and differentiation is believed to occur in niches in the marrow stromal microenvironment. Our recent observation that long-term in vitro human hematopoiesis requires a stromal heparan sulfate proteoglycan (HSPG) led us to hypothesize that such HSPG may orchestrate the formation of the stem cell niche. We compared the structure and function of HS from M2-10B4, a hematopoiesis-supportive cell line, with HS from a nonsupportive cell line, FHS-173-We. Long-term culture-initiating cell (LTC-IC) maintenance was enhanced by PG from supportive cells but not by PG from nonsupportive cells (P <.005). The supportive HS were significantly larger and more highly sulfated than the nonsupportive HS. Specifically, supportive HS contained higher 6-O-sulfation on the glucosamine residues. In agreement with these observations, purified 6-O-sulfated heparin and highly 6-O-sulfated bovine kidney HS similarly maintained LTC-IC. In contrast, completely desulfated heparin, N-sulfated heparin, and unmodified heparin did not support LTC-IC maintenance. Moreover, the supportive HS promoted LTC-IC maintenance but not differentiation of CD34(+)/HLA-DR- cells into colony-forming cells (CFCs) and mature blood cells. The supportive HS but not the nonsupportive HS bound both cytokines and matrix components critical for hematopoiesis, including interleukin-3 (IL-3), macrophage inflammatory protein-1 (MIP-1), and thrombospondin (TSP). Significantly more CD34(+) cells adhered directly to immobilized O-sulfated heparin than to N-sulfated or desulfated heparin. Thus, hematopoiesis-supportive stromal HSPG possessing large, highly 6-O-sulfated HS mediate the juxtaposition of hematopoietic progenitors with stromal cells, specific growth-promoting (IL-3) and growth-inhibitory (MIP-1 and platelet factor 4 [PF4]) cytokines, and extracellular matrix (ECM) proteins such as TSP. We conclude that the structural specificity of stromal HSPG that determines the selective colocalization of cytokines and ECM components leads to the formation of discrete niches, thereby orchestrating the controlled growth and differentiation of stem cells. These findings may have important implications for ex vivo expansion of and gene transfer into primitive hematopoietic progenitors.     

Engraftment of ex vivo expanded and cycling human cord blood hematopoietic progenitor cells in SCID mice

The ability of human hematopoietic cells to engraft SCID mice provides a useful model in which to study the efficiency of retroviral gene transfer and expression in primitive stem cells. In this regard, it is necessary to determine whether SCID mice can be engrafted by cycling human hematopoietic progenitor cells. Human cord blood cells from 12 different donors were cultured in vitro for 6 days with interleukin-3 and stem cell factor. Phenotypic analysis indicated that hematopoietic cells were induced to cycle and the number of progenitors was expanded, thus making them targets for retroviral gene transfer. The cells were then transferred to SCID mice. Human hematopoietic progenitor cell engraftment was assessed up to 7 weeks later by growth of human progenitor cells in soft agar. After in vitro culture under conditions used for retroviral gene transfer, human cord blood hematopoietic cells engrafted the bone marrow and spleen of SCID mice. Interestingly, cultured cord blood cells engrafted after intraperitoneal but not after intravenous injection. Furthermore, engraftment of cord blood cells was observed in mice receiving no irradiation before transfer of the human cells, suggesting that competition for space in the marrow is not a limiting factor when these cells have been cultured. Administration of human cytokines after transfer of human cord blood cells to SCID mice was also not required for engraftment. Thus, engraftment of SCID mice with human hematopoietic cells cultured under conditions suitable for gene transfer may provide an in vivo assay for gene transfer to early human hematopoietic progenitor cells.     

Experimental hypercholesterolemia induces ultrastructural changes in the elastic laminae of rabbit aortic valve

The engraftment of hematopoietic stem and progenitor cells in lethally irradiated mice was evaluated following transplants of enriched hematopoietic cell populations which were defined by surface antigen and rhodamine-123 staining. Phenotypically defined long-term repopulating stem cells, short-term pluripotent progenitors, and committed myeloerythroid progenitors all rapidly reconstituted splenic cellularity and peripheral red blood cells after transplant into myeloablated animals. In contrast, marrow cellularity was reconstituted only after transplant of long-term repopulating stem cells. In addition, peripheral blood platelet and lymphocyte counts increased only after transplantation of the long-term repopulating population. Transplantation of highly enriched multipotent progenitors resulted in a transient increase in peripheral blood myeloid cells that occurred with kinetics similar to that seen after transplant of the primitive stem cell population. Erythroid reconstitution was similar in all groups, suggesting that the effect of myeloerythroid progenitor cells in mouse marrow transplants is primarily on reconstitution of the erythroid lineage due to splenic hematopoiesis. Collectively, these results suggest that the cells which function to rapidly reconstitute the nucleated blood cells in a transplant setting are more closely related to primitive, marrow-homing stem cells than to committed progenitor cells.