Totipotent Hematopoietic Stem Cells Research Articles

Gene therapy of primary immunodeficiencies.

Gene therapy is often considered the ultimate treatment with its obvious potential advantage, the correction of genetic diseases at the molecular level, as opposed to merely treating the symptoms caused by genetic aberrations. This vision has spurred great interest in the development and clinical application of gene transfer-based therapeutic approaches. Moreover, recent progress in biotechnology and recombinant DNA techniques has greatly improved our understanding of the molecular basis of inherited diseases, providing the knowledge base necessary to attempt their treatment by gene transfer. In the last decade in particular, genetic defects underlying most of the primary immunodeficiency diseases (PIDD) have been identified (see Table 1), opening the way to the possibility of genetic correction. Theoretically, all PIDD that are curable by allogeneic bone marrow transplantation (BMT) are candidates for ex vivo gene therapy. However, because extensive knowledge of the function(s) and regulation of the involved gene is a clear prerequisite to the rational development of new gene-based therapies, diseases caused by mutations of genes whose function is still unclear (e.g. Xlinked agammaglobulinemia, Wiskott-Aldrich syndrome, and Chediak-Higashi syndrome) must await further studies before clinical gene transfer protocols for affected patients can be proposed. Gene transfer into the totipotent hematopoietic stem cell would seem to be the most logical strategy for treating most PIDD. Targeting the stem cell, however, has proven to be a very difficult task for a series of reasons. These include the lack of precise information about the phenotypic characteristics of the "true" self-renewing lymphohematopoietic stem cell capable of reconstituting the immune system and the very low gene transfer efficiency into such a functional unit that is achievable with current techniques. The results of the extensive clinical experience with BMT as treatment of severe combined immunodeficiencies (SCID) and other PIDD have indicated that the pres-

Thrombopoietic potential and serial repopulating ability of murine hematopoietic stem cells constitutively expressing interleukin 11.

Based on transplantation studies with bone marrow cultured under various conditions, a role of interleukin 11 (IL-11) in the self-renewal and/or the differentiation commitment of hematopoietic stem cells has been indicated. To better evaluate the in vivo effects of IL-11 on stem/progenitor cell biology, lethally irradiated mice were serially transplanted with bone marrow cells transduced with a defective retrovirus, termed MSCV-mIL-11, carrying the murine IL-11 (mIL-11) cDNA and the bacterial neomycin phosphotransferase (neo) gene. High serum levels (i.e., > 1 ng/ml) of mIL-11 in all (20/20) primary and 86% (12/14) of secondary long-term reconstituted mice, as well as 86% (12/14) of tertiary recipients examined at 6 weeks posttransplant, demonstrated persistence of vector expression subsequent to transduction of bone marrow precursors functionally definable as totipotent hematopoietic stem cells. In agreement with results obtained with human IL-11 in other myeloablation models, ectopic mIL-11 expression accelerated recovery of platelets, neutrophils, and, to some extent, total leukocytes while preferentially increasing peripheral platelet counts in fully reconstituted mice. When analyzed 5 months posttransplant, tertiary MSCV-mIL-11 recipients had a significantly greater percentage of G418-resistant colony-forming cells in their bone marrow compared with control MSCV animals. Collectively, these data show that persistent stimulation of platelet production by IL-11 is not detrimental to stem cell repopulating ability; rather, they suggest that IL-11 expression in vivo may have resulted in enhanced maintenance of the most primitive hematopoietic stem cell compartment. The prolonged expression achieved by the MSCV retroviral vector, despite the presence of a selectable marker, contrasts with the frequent transcriptional extinction observed with other retroviral vectors carrying two genes. These findings have potentially important implications for clinical bone marrow transplantation and gene therapy of the hematopoietic system.

Proliferation of multipotent hematopoietic cells controlled by a truncated erythropoietin receptor transgene.

The long-term efficacy of gene therapy using bone marrow transplantation requires the engraftment of genetically altered totipotent hematopoietic stem cells (THSCs). Ex vivo expansion of corrected THSCs is one way to increase the efficiency of the procedure. Similarly, selective in vivo expansion of the therapeutic THSCs rather than the endogenous THSCs could favor the transplant. To test whether a conferred proliferative advantage gene can facilitate the in vitro and in vivo expansion of hematopoietic stem cells, we have generated transgenic mice expressing a truncated receptor for the growth factor erythropoietin. These mice are phenotypically normal, but when treated in vivo with exogenous erythropoietin they exhibit a marked increase in multipotent, clonogenic hematopoietic cells [colony-forming units in the spleen (CFU-S) and CFUs that give rise to granulocytes, erythroid cells, macrophages, and megakaryocytes within the same colony (CFU-GEMM)] in comparison with the wild-type mice. In addition, long-term in vitro culture of tEpoR transgenic bone marrow in the presence of erythropoietin induces exponential expansion of trilineage hematopoietic stem cells not seen with wild-type bone marrow. Thus, the truncated erythropoietin receptor gene shows promise as a means for obtaining cytokine-inducible hematopoietic stem cell proliferation to facilitate the direct targeting of THSCs and to provide a competitive repopulation advantage for transplanted therapeutic stem cells.

Natural History of Paroxysmal Nocturnal Hemoglobinuria

Paroxysmal nocturnal hemoglobinuria (PNH), which is characterized by intravascular hemolysis and venous thrombosis, is an acquired clonal disorder associated with a somatic mutation in a totipotent hematopoietic stem cell. An understanding of the natural history of PNH is essential to improve therapy. We have followed a group of 80 consecutive patients with PNH who were referred to Hammersmith Hospital, London, between 1940 and 1970. They were treated with supportive measures, such as oral anticoagulant therapy after established thromboses, and transfusions. The median age of the patients at the time of diagnosis was 42 years (range, 16 to 75), and the median survival after diagnosis was 10 years, with 22 patients (28 percent) surviving for 25 years. Sixty patients have died; 28 of the 48 patients for whom the cause of death is known died from either venous thrombosis or hemorrhage. Thirty-one patients (39 percent) had one or more episodes of venous thrombosis during their illness. Of the 35 patients who survived for 10 years or more, 12 had a spontaneous clinical recovery. No PNH-affected cells were found among the erythrocytes or neutrophils of the patients in prolonged remission, but a few PNH-affected lymphocytes were detectable in three of the four patients tested. Leukemia did not develop in any of the patients. PNH is a chronic disorder that curtails life. A spontaneous long-term remission can occur, which must be taken into account when considering potentially dangerous treatments, such as bone marrow transplantation. Platelet transfusions should be given, as appropriate, and long-term anticoagulation therapy should be considered for all patients.

Characterization and Purification of a Primitive Hematopoietic Cell Type in Adult Mouse Marrow Capable of Lymphomyeloid Differentiation in Long-Term Marrow “Switch” Cultures

In this report, we describe a modification of the assay for long-term culture-initiating cells (LTC-IC) that allows a subset of murine LTC-IC (designated as LTC-ICML) to express both their myeloid (M) and lymphoid (L) differentiative potentials in vitro. The modified assay involves culturing test cells at limiting dilutions on irradiated mouse marrow feeder layers for an initial 4 weeks under conditions that support myelopoiesis and then for an additional week under conditions permissive for B-lymphopoiesis. All of the clonogenic pre-B progenitors (colony-forming unit [CFU] pre-B) detected in such postswitch LTC appear to be the progeny of uncommitted cells present in the original cell suspension because exposure of lymphoid-restricted progenitors to myeloid LTC conditions for > or = 7 days was found to irreversibly terminate CFU-pre-B production and, in cultures initiated with limiting numbers of input cells (no progenitors of any type detected in > 70% of cultures 1 week after the switch), the presence of CFU-pre-B was tightly associated with the presence of myeloid clonogenic cells, regardless of the purity of the input population. Limiting dilution analysis of the proportion of negative cultures measured for different numbers of input cells showed the frequency of LTC-ICML in normal adult mouse marrow to be 1 per 5 x 10(5) cells with an enrichment of approximately 500-fold in the Sca-1+ Lin-WGA+ fraction, as was also found for competitive in vivo repopulating units (CRU) and conventionally defined LTC-IC. LTC-ICML also exhibited the same resistance to treatment in vivo with 5-fluorouracil (5-FU) as CRU and LTC-IC, thereby distinguishing these three populations from the great majority of both in vitro clonogenic cells and day 12 CFU-S. The ability to quantitate cells with dual lymphoid and myeloid differentiation potentials in vitro, without the need for their prior purification, should facilitate studies of totipotent hematopoietic stem cell regulation.

Overexpression of HOXB4 in hematopoietic cells causes the selective expansion of more primitive populations in vitro and in vivo.

Hox genes were first recognized for their role in embryonic development and may also play important lineage-specific functions in a variety of somatic tissues including the hematopoietic system. We have recently shown that certain members of the Hox A and B clusters, such as HOXB3 and HOXB4, are preferentially expressed in subpopulations of human bone marrow that are highly enriched for the most primitive hematopoietic cell types. To assess the role these genes may play in regulating the proliferation and/or differentiation of such cells, we engineered the overexpression of HOXB4 in murine bone marrow cells by retroviral gene transfer and analyzed subsequent effects on the behavior of various hematopoietic stem and progenitor cell populations both in vitro and in vivo. Serial transplantation studies revealed a greatly enhanced ability of HOXB4-transduced bone marrow cells to regenerate the most primitive hematopoietic stem cell compartment resulting in 50-fold higher numbers of transplantable totipotent hematopoietic stem cells in primary and secondary recipients, compared with serially passaged neo-infected control cells. This heightened expansion in vivo of HOXB4-transduced hematopoietic stem cells was not accompanied by identifiable anomalies in the peripheral blood of these mice. Enhanced proliferation in vitro of day-12 CFU-S and clonogenic progenitors was also documented. These results indicate HOXB4 to be an important regulator of very early but not late hematopoietic cell proliferation and suggest a new approach to the controlled amplification of genetically modified hematopoietic stem cell populations.

Highly sensitive polymerase chain reaction methods show the frequent survival of residual recipient multipotent progenitors after non-T-cell- depleted bone marrow transplantation

Twenty-four male patients grafted for various pathologies with the marrow of a female donor and presenting a complete donor-type hematopoiesis when analyzed by polymerase chain reaction (PCR) amplification of minisatellite sequences 33.6.3 and MS51 (0.1% to 1% sensitivity) were studied by the highly sensitive technique of PCR amplification of the Y-chromosome-specific DYZ1 sequence (0.01% sensitivity). Residual recipient male cells were detected in all peripheral blood samples collected within 1 year posttransplantation. These residual cells were present in both the lymphocyte and polymorphonuclear cell fractions when such a separation was performed by Ficoll gradient centrifugation and, for samples of 13 of 15 patients, at comparable levels in both fractions. In 3 samples collected from 3 patients 4 months or more posttransplantation, residual recipient cells were detected in the polymorphonuclear cell fraction but were present at a lower level or were undetectable in the lymphocyte fraction. These cells are of hematopoietic origin because they were detected at equivalent levels in whole blood and in B and T lymphocytes sorted with antibody-coated magnetic beads. They were not detected in samples collected more than 15 months posttransplantation for 6 of 7 patients. The persistence of residual recipient cells within 1 year posttransplantation is not restricted to male patients receiving a transplant from a female donor because they were also detected in 2 female patients using an allele-specific amplification method for the thyroid peroxydase gene that also has a high sensitivity (0.01%). Our results indicate that at least residual recipient myeloid progenitors and possibly totipotent hematopoietic stem cells may survive intensive pretransplant conditioning regimen and support a transient residual hematopoiesis of the host posttransplantation.

Highly Sensitive Polymerase Chain Reaction Methods Show the Frequent Survival of Residual Recipient Multipotent Progenitors After Non–T-Cell–Depleted Bone Marrow Transplantation

Twenty-four male patients grafted for various pathologies with the marrow of a female donor and presenting a complete donor-type hematopoiesis when analyzed by polymerase chain reaction (PCR) amplification of minisatellite sequences 33.6.3 and MS51 (0.1% to 1% sensitivity) were studied by the highly sensitive technique of PCR amplification of the Y-chromosome-specific DYZ1 sequence (0.01% sensitivity). Residual recipient male cells were detected in all peripheral blood samples collected within 1 year posttransplantation. These residual cells were present in both the lymphocyte and polymorphonuclear cell fractions when such a separation was performed by Ficoll gradient centrifugation and, for samples of 13 of 15 patients, at comparable levels in both fractions. In 3 samples collected from 3 patients 4 months or more posttransplantation, residual recipient cells were detected in the polymorphonuclear cell fraction but were present at a lower level or were undetectable in the lymphocyte fraction. These cells are of hematopoietic origin because they were detected at equivalent levels in whole blood and in B and T lymphocytes sorted with antibody-coated magnetic beads. They were not detected in samples collected more than 15 months posttransplantation for 6 of 7 patients. The persistence of residual recipient cells within 1 year posttransplantation is not restricted to male patients receiving a transplant from a female donor because they were also detected in 2 female patients using an allele-specific amplification method for the thyroid peroxydase gene that also has a high sensitivity (0.01%). Our results indicate that at least residual recipient myeloid progenitors and possibly totipotent hematopoietic stem cells may survive intensive pretransplant conditioning regimen and support a transient residual hematopoiesis of the host posttransplantation.

Efficient gene transfer into nondividing cells by adeno-associated virus-based vectors.

Gene transfer vectors based on adeno-associated virus (AAV) are emerging as highly promising for use in human gene therapy by virtue of their characteristics of wide host range, high transduction efficiencies, and lack of cytopathogenicity. To better define the biology of AAV-mediated gene transfer, we tested the ability of an AAV vector to efficiently introduce transgenes into nonproliferating cell populations. Cells were induced into a nonproliferative state by treatment with the DNA synthesis inhibitors fluorodeoxyuridine and aphidicolin or by contact inhibition induced by confluence and serum starvation. Cells in logarithmic growth or DNA synthesis arrest were transduced with vCWR:beta gal, an AAV-based vector encoding beta-galactosidase under Rous sarcoma virus long terminal repeat promoter control. Under each condition tested, vCWR:beta Gal expression in nondividing cells was at least equivalent to that in actively proliferating cells, suggesting that mechanisms for virus attachment, nuclear transport, virion uncoating, and perhaps some limited second-strand synthesis of AAV vectors were present in nondividing cells. Southern hybridization analysis of vector sequences from cells transduced while in DNA synthetic arrest and expanded after release of the block confirmed ultimate integration of the vector genome into cellular chromosomal DNA. These findings may provide the basis for the use of AAV-based vectors for gene transfer into quiescent cell populations such as totipotent hematopoietic stem cells.

Clinical Effects of Biologic Response Modifiers

The clinical use of the biologic response modifiers filgrastim, sargramostim, and regramostim is reviewed. All circulating blood cells are derived from totipotent hematopoietic stem cells. Various biologic response modifiers, including lymphokines and colony-stimulating factors, regulate and activate the lymphoid and myeloid cells of the blood. One of the more important types of blood cell for fighting infection is the neutrophil. Patients with low neutrophil concentrations are at high risk of developing neutropenic fevers and infections. The colony-stimulating factors filgrastim, sargramostim, and regramostim increase the production of circulating neutrophils, and this action is clinically useful in patients undergoing myelosuppressive antineoplastic therapy or bone marrow transplantation and in patients with the acquired immunodeficiency syndrome. Clinical studies of these agents in comparison with antimicrobial prophylaxis or placebo have shown a decreased rate of neutropenic-associated hospitalizations and infections. These agents are also under study for dose intensification of antineoplastics in patients with various solid tumors and for augmenting patient responses to antimicrobial therapy in situations where there is high risk of morbidity and mortality. Sargramostim and regramostim are both granulocyte-macrophage colony-stimulating factors that differ in their degree of glycosylation and source of production, and at high doses they can cause life-threatening adverse effects because they stimulate the production of a broad range of leukocytes. Filgrastim, which stimulates only the production of neutrophils, has been better tolerated, especially at higher doses. Biologic response modifiers hold much promise for improving therapy of certain clinical conditions by decreasing myelosuppressive complications and enhancing responses to other drugs.(ABSTRACT TRUNCATED AT 250 WORDS)

Proliferation of totipotent hematopoietic stem cells in vitro with retention of long-term competitive in vivo reconstituting ability.

Marrow cells from male mice pretreated with 5-fluorouracil were infected with helper-free neomycin-resistant (neor) recombinant retrovirus and then used to initiate long-term cultures (LTC) on irradiated adherent marrow feeder layers. Four weeks later LTC cells were harvested and injected into lethally irradiated female recipients either alone or together with 2 x 10(5) female marrow cells with selectively compromised long-term repopulating potential to assay for totipotent and competitive repopulating units (CRU), respectively. A total of 46 unique clones were detected in recipients 5 wk to 7 mo after transplant. Half of these clones (22 of 46) included both lymphoid and myeloid progeny. Eight of the 22 lympho-myeloid clones were represented in multiple recipients, in some cases after injection of limiting numbers of CRU, thus indicating repopulation from sibling totipotent stem cells generated during the initial 4-wk period in LTC. Serial analysis of cells released into the nonadherent fraction of LTC for up to 7 wk provided additional evidence of the continuing proliferation in LTC of totipotent stem cells with long-term repopulating potential. The frequency of CRU determined from limiting-dilution analyses of LTC-derived cells was the same for recipients analyzed at 5 wk or 7 mo after transplantation and was also the same whether marrow or thymus repopulation was assessed. These assays showed that concurrent with the expansion of some totipotent cells revealed by retroviral marking, there was a slow but net 6.5-fold decrease in total CRU numbers after 4 wk in LTC. These results show the capacity of some totipotent hematopoietic stem cells to be maintained and amplified over extensive time periods in vitro without diminution of their long-term in vivo repopulating potential. These results also set the stage for analogous studies of human stem cell selection and expansion in vitro, which may be important for future gene therapy protocols.

Retroviral vector-mediated gene transfer into hematopoietic cells: prospects and issues.

Gene therapy is a developing technology that may allow the treatment of a variety of congenital and acquired genetic disorders as well as infectious diseases through the introduction of exogenous genetic material into relevant cellular populations. Currently, the most effective method for gene transfer into cells of the hematopoietic system is with retroviral vectors. Appropriate cellular targets for gene transfer include totipotent hematopoietic stem cells as well as long-lived lineage committed cells such as T lymphocytes. Although retroviral vector-mediated gene transfer into totipotent stem cells and subsequent long-term expression of transduced genetic material in stem cell progeny has been observed in murine bone marrow transplantation experiments, similar observations have not been made in clinically relevant large-animal models. A number of recent advances in gene delivery systems, purification of stem cells, defining extramedullary sources of stem cells, characterizing the biologic processes that regulate the proliferation and developmental potential of stem cells, and construction of more effective models for assessing stem cells, may result in improvements in gene transfer into large animal and human totipotent stem cells.

Temporal replacement of donor erythrocytes and leukocytes in nonanemic W44J/W44J and severely anemic W/Wv mice

The dominant white spotting, W, locus in the mouse encodes Kit, a receptor molecule with cytosolic tyrosine kinase activity. Mutations in Kit deplete hematopoietic cells by an as yet unknown mechanism, but one that presumably affects the early progenitors of all cell lineages. To examine cell lineage-specific changes caused by different W mutations, we injected genetically marked normal marrow cells into mutant mice and monitored repopulation kinetics. In the present report, we compare repopulation of the various peripheral blood cells in nonanemic W44J/W44J and severely anemic W/Wv mice administered increasing increments of donor cells. At all doses of cells tested, donor erythrocyte repopulation precedes leukocyte repopulation regardless of the recipient phenotype. There is, in fact, little difference in the rate or extent of nonerythroid repopulation in W44J/W44J mice injected with between 6 x 10(6) and 2 x 10(7) donor cells. The fact that donor cells rapidly replace erythrocytes, even in the nonanemic W44J/W44J host, while other cell lineages become donor type more slowly provides further evidence that mutations at the W locus are especially damaging to erythrocyte progenitors. We suggest that host nonerythroid hematopoietic cells compete with normal cells, probably at the level of early progenitors rather than at the level of the totipotent hematopoietic stem cell. The fact that successively higher doses of donor cells do not markedly alter nonerythroid repopulation kinetics implies that it may be possible to maximize autologous therapeutic marrow transplantation.

Temporal Replacement of Donor Erythrocytes and Leukocytes in Nonanemic W44J/W44J and Severely Anemic W/Wv Mice

Abstract The dominant white spotting, W, locus in the mouse encodes Kit, a receptor molecule with cytosolic tyrosine kinase activity. Mutations in Kit deplete hematopoietic cells by an as yet unknown mechanism, but one that presumably affects the early progenitors of all cell lineages. To examine cell lineage-specific changes caused by different W mutations, we injected genetically marked normal marrow cells into mutant mice and monitored repopulation kinetics. In the present report, we compare repopulation of the various peripheral blood cells in nonanemic W44J/W44J and severely anemic W/Wv mice administered increasing increments of donor cells. At all doses of cells tested, donor erythrocyte repopulation precedes leukocyte repopulation regardless of the recipient phenotype. There is, in fact, little difference in the rate or extent of nonerythroid repopulation in W44J/W44J mice injected with between 6 x 10(6) and 2 x 10(7) donor cells. The fact that donor cells rapidly replace erythrocytes, even in the nonanemic W44J/W44J host, while other cell lineages become donor type more slowly provides further evidence that mutations at the W locus are especially damaging to erythrocyte progenitors. We suggest that host nonerythroid hematopoietic cells compete with normal cells, probably at the level of early progenitors rather than at the level of the totipotent hematopoietic stem cell. The fact that successively higher doses of donor cells do not markedly alter nonerythroid repopulation kinetics implies that it may be possible to maximize autologous therapeutic marrow transplantation.

Molecular Analysis of Primitive Hematopoietic Cell Proliferation Control Mechanismsa

Cells at two distinct early stages in the development of mature human blood cells from primitive totipotent hematopoietic stem cells can now be defined and quantitated by separate in vitro assays. Current evidence suggests that most, if not all, colony-forming cells--that is, cells that give rise to colonies of mature progeny within one to three weeks in semisolid culture systems, represent an intermediate stage of hematopoietic progenitor. These cells are not self-sustaining; if they are used to initiate hematopoiesis on competent marrow stromal layers, they rapidly disappear as they differentiate or die. However, clonogenic cells can be generated in such cultures from another cell type over a period of four to eight weeks. We have, therefore, assigned the term long-term culture initiating cell (LTC-IC) to this latter type of clonogenic precursor cell. The production and differentiation of cells in both of these compartments in LTC are dependent on, and regulated by, nonhematopoietic "stromal" cells that form a heterogeneous adherent layer in which close-range interactions with hematopoietic cells take place. The use of separate endpoints to monitor the maintenance, differentiation, and reversible activation or arrest of cycling of these cells has recently revealed different molecular mechanisms regulating their respective functions. However, an important common feature appears to be the relative local concentration of positive and negative regulators to which the target hematopoietic cell is exposed. Both gene expression and growth factor release measurements as well as results obtained using genetically engineered stroma and repeated soluble growth factor addition implicate G-CSF as an endogenous positive regulator of primitive hematopoietic cells. Similarly, gene expression, factor production, factor addition, and neutralizing antibody experiments implicate TGF-beta as an endogenous inhibitor of primitive hematopoietic cells.

A receptor tyrosine kinase specific to hematopoietic stem and progenitor cell-enriched populations

To elucidate the molecular biology of the hematopoietic stem cell, we have begun to isolate genes from murine cell populations enriched in stem cell activity. One such cDNA encodes a novel receptor tyrosine kinase, designated fetal liver kinase-2 or flk-2, which is related to the W locus gene product c- kit. Expression analyses suggest an extremely restricted distribution of flk-2. It is expressed in populations enriched for stem cells and primitive uncommitted progenitors, and is absent in populations containing more mature cells. Therefore, this receptor may be a key signal transducing component in the totipotent hematopoietic stem cell and its immediate self-renewing progeny.

Expansion in vitro of retrovirally marked totipotent hematopoietic stem cells

A large number of biologic, technological, and clinical studies await the development of procedures that will allow totipotent hematopoietic stem cells to be expanded in vitro. Previous work has suggested that hematopoiesis can be reconstituted using transplants of cells from long-term marrow cultures. We have used retrovirus mediated gene transfer to demonstrate that marked totipotent hematopoietic stem cells are both maintained and can be amplified in such cultures, and then subsequently regenerate and sustain lympho-myeloid hematopoiesis in irradiated recipients. Marrow cells from 5-fluorouracil-treated male mice were infected with a recombinant virus carrying the neomycin resistence gene and seeded onto irradiated adherent layers of pre-established, long-term marrow cultures of female origin. At 4 weeks, cells from individual cultures were transplanted into single or multiple female recipients. Southern blot analysis of hematopoietic tissue 45 days posttransplantation showed retrovirally marked clones common to lymphoid and myeloid tissues in 14 of 23 mice examined. Strikingly, for 3 of 4 long-term cultures, multiple recipients of cells from a single flask showed marrow and thymus repopulation with the same unique retrovirally marked clone. These results establish the feasibility of retroviral-marking techniques to demonstrate the maintenance of totipotent lympho-myeloid stem cells for at least 4 weeks in the long-term marrow culture system and provide the first evidence of their proliferation in vitro. Therefore, such cultures may serve as a starting point for identifying factors that stimulate totipotent hematopoietic stem cell expansion.

Expansion In Vitro of Retrovirally Marked Totipotent Hematopoietic Stem Cells

Abstract A large number of biologic, technological, and clinical studies await the development of procedures that will allow totipotent hematopoietic stem cells to be expanded in vitro. Previous work has suggested that hematopoiesis can be reconstituted using transplants of cells from long- term marrow cultures. We have used retrovirus mediated gene transfer to demonstrate that marked totipotent hematopoietic stem cells are both maintained and can be amplified in such cultures, and then subsequently regenerate and sustain lympho-myeloid hematopoiesis in irradiated recipients. Marrow cells from 5-fluorouracil-treated male mice were infected with a recombinant virus carrying the neomycin resistence gene and seeded onto irradiated adherent layers of pre-established, long- term marrow cultures of female origin. At 4 weeks, cells from individual cultures were transplanted into single or multiple female recipients. Southern blot analysis of hematopoietic tissue 45 days posttransplantation showed retrovirally marked clones common to lymphoid and myeloid tissues in 14 of 23 mice examined. Strikingly, for 3 of 4 long-term cultures, multiple recipients of cells from a single flask showed marrow and thymus repopulation with the same unique retrovirally marked clone. These results establish the feasibility of retroviral-marking techniques to demonstrate the maintenance of totipotent lympho-myeloid stem cells for at least 4 weeks in the long- term marrow culture system and provide the first evidence of their proliferation in vitro. Therefore, such cultures may serve as a starting point for identifying factors that stimulate totipotent hematopoietic stem cell expansion.

Long- and short-lived murine hematopoietic stem cell clones individually identified with retroviral integration markers

The proliferative longevity of totipotent hematopoietic stem cells (THSC) is a limiting factor in normal hematopoiesis and in therapy by cell- or gene-replacement, but has not yet been ascertained. We have followed the long-term fate of individual clones of mouse THSC from fetal liver or adult bone marrow, after labeling in culture, followed by engraftment and serial transplantation in unirradiated W/Wv-C57BL/6 hosts. The ancestor cell of each clone and its mitotic progeny were uniquely identifiable retrospectively by the DNA integration pattern experimentally produced by replication-incompetent recombinant murine retroviruses. These viruses provided physiologically neutral markers. The marked clones proved to be derived from THSC, based on their contributions to a wide array of myeloid and lymphoid blood lineages in the hosts. The label also identified the target cells as the population displaying clonal succession. The various labeled stem cell clones proliferated for substantially different periods of time. The longest observed clone endured, after the original cell was marked, for at least 2 1/2 years--the equivalent of a mouse's lifetime. However, the results suggest that THSC clones are not all long-lived and that even the longest-lived ones may not be potentially immortal. Thus, the unpredictable lifespan of any given THSC clone indicates the desirability of introducing multiple clones in therapeutic transplants.

Cellular and developmental properties of fetal hematopoietic stem cells

We have characterized the fetal totipotent hematopoietic stem cell using a novel strategy that integrates physical analysis of cell properties and genetic analysis of in vivo developmental behavior. This approach allows the simultaneous isolation and in vivo characterization of any stem cell population. Using this procedure we demonstrate that a cell surface marker, recognized by monoclonal antibody AA4.1, defines 0.5%-1.0% of fetal liver tissue that contains the entire hierarchy of primitive hematopoietic cells. The AA4.1+ subpopulation includes multipotential in vitro progenitors, CFU-S cells, and lymphoid-myeloid stem cells that function to yield permanent and oligoclonal blood systems. Further fractionation of these cells by analysis of density, fibronectin binding, and surface antigen distribution has defined 0.1%-0.2% of fetal liver that contains the totipotent stem cell.