Progress to uncover the molecular and cellular regulators that govern human hematopoietic stem ce... more Progress to uncover the molecular and cellular regulators that govern human hematopoietic stem cell (HSC) fate has been impeded by an inability to obtain highly purified fractions of HSCs. We report that the rhodamine 123 (Rho 123) dye effluxing fraction of the Lin ؊ CD34 ؉ CD38 ؊ population contains SCID-repopulating cells (SRCs) capable of long-term repopulation in primary nono-bese diabetic/severe combined immunodeficient (NOD/SCID) mice. Purification based on Rho uptake led to a 4-fold enrichment of SRCs in the Lin ؊ CD34 ؉ CD38 ؊ fraction, with a frequency of 1 SRC in 30 Lin ؊ CD34 ؉ CD38 ؊ Rho lo cells. The Lin ؊ CD34 ؉ CD38 ؊ Rho lo fraction also possesses long-term selfrenewal capacity as measured by serial transplantation totaling more than 20 weeks. We conclude that Rho dye efflux provides an additional means of purifying human HSCs in the quest to achieve homogeneous populations of primitive cells for both experimental and therapeutic applications. (Blood.
Ł € Hematopoiesis and Stem Cells (3132 articles)Ł € Gene Therapy (524 articles)Articles on simila... more Ł € Hematopoiesis and Stem Cells (3132 articles)Ł € Gene Therapy (524 articles)Articles on similar topics can be found in the following Blood collectionshttp://bloodjournal.hematologylibrary.org/site/misc/rights.xhtml#repub_requests Information about reproducing this article in parts or in its entirety may be found online at:http://bloodjournal.hematologylibrary.org/site/misc/rights.xhtml#reprints Information about ordering reprints may be found online at:http://bloodjournal.hematologylibrary.org/site/subscriptions/index.xhtml Information about subscriptions and ASH membership may be found online at:
Leukemia in humans arises from the multistep accumulation of mutations. However, the identity of ... more Leukemia in humans arises from the multistep accumulation of mutations. However, the identity of the cell of origin, the nature of the first genetic lesion and the order of subsequent mutations remain poorly understood, as most cases of de novo acute myeloid leukemia (AML) are diagnosed without prior observation of a pre-leukemic clonal expansion. As part of studies to examine intra-tumoral genetic heterogeneity in AML, we carried out deep targeted sequencing (read depth 250×) of 101 commonly mutated leukemia genes on samples from 12 patients at diagnosis. Normal T-cells from each sample were expanded in vitro to provide a non-leukemic hematopoietic tissue for comparison. In 3 of 4 patients, we unexpectedly identified DNMT3a mutation not only in AML cells but also in T-cells at a low allele frequency (1-20%). Other genetic alterations such as NPM1 mutation (mutNPM1) were found only in AML cells and not in T-cells, ruling out contaminating AML cells as the source of the DNMT3a signal...
In acute myeloid leukaemia, long-term survival is poor as most patients relapse despite achieving... more In acute myeloid leukaemia, long-term survival is poor as most patients relapse despite achieving remission. Historically, the failure of therapy has been thought to be due to mutations that produce drug resistance, possibly arising as a consequence of the mutagenic properties of chemotherapy drugs. However, other lines of evidence have pointed to the pre-existence of drug-resistant cells. For example, deep sequencing of paired diagnosis and relapse acute myeloid leukaemia samples has provided direct evidence that relapse in some cases is generated from minor genetic subclones present at diagnosis that survive chemotherapy, suggesting that resistant cells are generated by evolutionary processes before treatment and are selected by therapy. Nevertheless, the mechanisms of therapy failure and capacity for leukaemic regeneration remain obscure, as sequence analysis alone does not provide insight into the cell types that are fated to drive relapse. Although leukaemia stem cells have been linked to relapse owing to their dormancy and self-renewal properties, and leukaemia stem cell gene expression signatures are highly predictive of therapy failure, experimental studies have been primarily correlative and a role for leukaemia stem cells in acute myeloid leukaemia relapse has not been directly proved. Here, through combined genetic and functional analysis of purified subpopulations and xenografts from paired diagnosis/relapse samples, we identify therapy-resistant cells already present at diagnosis and two major patterns of relapse. In some cases, relapse originated from rare leukaemia stem cells with a haematopoietic stem/progenitor cell phenotype, while in other instances relapse developed from larger subclones of immunophenotypically committed leukaemia cells that retained strong stemness transcriptional signatures. The identification of distinct patterns of relapse should lead to improved methods for disease management and monitoring in acute myeloid leukaemia. Moreover, the shared functional and transcriptional stemness properties that underlie both cellular origins of relapse emphasize the importance of developing new therapeutic approaches that target stemness to prevent relapse.
Based on initial observations of human CD34+ Thy-1+ cells and long-term culture-initiating cells ... more Based on initial observations of human CD34+ Thy-1+ cells and long-term culture-initiating cells (LTC-IC) in the bone marrow of some sublethally irradiated severe combined immunodeficient (SCID) mice transplanted intravenously with normal human marrow cells, and the subsequent finding that the NOD/LtSz-scid/scid (NOD/SCID) mouse supports higher levels of human cell engraftment, we undertook a series of time course experiments to examine posttransplant changes in the number, tissue distribution, cycling activity, and in vivo differentiation pattern of various human hematopoietic progenitor cell populations in this latter mouse model. These studies showed typical rapid posttransplant recovery curves for human CD34- CD19+ (B-lineage) cells, CD34+ granulopoietic, erythroid, and multilineage colony-forming cells (CFC), LTC-IC, and CD34+ Thy-1+ cells from a small initial population representing <0.1% of the original transplant. The most primitive human cell populations reached maximum ...
The ability to transfer new genetic material into human hematopoietic cells provides the foundati... more The ability to transfer new genetic material into human hematopoietic cells provides the foundation for characterizing the organization and developmental program of human hematopoietic stem cells. It also provides a valuable model in which to test gene transfer and long-term expression in human hematopoietic cells as a prelude to human gene therapy. At the present time such studies are limited by the absence of in vivo assays for human stem cells, although recent descriptions of the engraftment of human hematopoietic cells in immune-deficient mice may provide the basis for such an assay. This study focuses on the establishment of conditions required for high efficiency retrovirus-mediated gene transfer into human hematopoietic progenitors that can be assayed in vitro in short-term colony assays and in vivo in immune-deficient mice. Here we report that a 24-hour preincubation of human bone marrow in 5637-conditioned medium, before infection, increases gene transfer efficiency into in...
Fanconi anemia (FA) is a pleiotropic inherited disease that causes bone marrow failure in childre... more Fanconi anemia (FA) is a pleiotropic inherited disease that causes bone marrow failure in children. However, the specific involvement of FA genes in hematopoiesis and their relation to bone marrow (BM) failure is still unclear. The increased sensitivity of FA cells to DNA cross-linking agents such as mitomycin C (MMC) and diepoxybutane (DEB), including the induction of chromosomal aberrations and delay in the G2 phase of the cell cycle, have suggested a role for the FA genes in DNA repair, cell cycle regulation, and apoptosis. We previously reported the cloning of the FA group C gene (FAC) and the generation of a Fac mouse model. Surprisingly, the Fac -/- mice did not show any of the hematologic defects found in FA patients. To better understand the relationship of FA gene functions to BM failure, we have analyzed the in vivo effect of an FA-specific DNA damaging agent in Fac -/- mice. The mice were found to be highly sensitive to DNA cross-linking agents; acute exposure to MMC prod...
We have previously reported the development of in vivo functional assays for primitive human hema... more We have previously reported the development of in vivo functional assays for primitive human hematopoietic cells based on their ability to repopulate the bone marrow (BM) of severe combined immunodeficient (SCID) and nonobese diabetic/SCID (NOD/SCID) mice following intravenous transplantation. Accumulated data from gene marking and cell purification experiments indicate that the engrafting cells (defined as SCID-repopulating cells or SRC) are biologically distinct from and more primitive than most cells that can be assayed in vitro. Here we demonstrate through limiting dilution analysis that the NOD/SCID xenotransplant model provides a quantitative assay for SRC. Using this assay, the frequency of SRC in cord blood (CB) was found to be 1 in 9.3 x 10(5) cells. This was significantly higher than the frequency of 1 SRC in 3.0 x 10(6) adult BM cells or 1 in 6.0 x 10(6) mobilized peripheral blood (PB) cells from normal donors. Mice transplanted with limiting numbers of SRC were engrafted...
A major problem hampering effective stem cell-based therapies is the absence of a clear understan... more A major problem hampering effective stem cell-based therapies is the absence of a clear understanding of the human hematopoietic stem cell (HSC) pool composition. The severe combined immunodeficiency (SCID) repopulating cell (SRC) xenotransplant assay system provides a powerful tool for characterizing the frequency, cell surface markers, cell cycle status, homing and response to cytokine stimulation of human HSCs 1-3. Clonal tracking of retrovirally transduced SRCs and transplantation of specific subpopulations revealed SRC classes with distinct repopulation potentials 4-7. However, all HSC repopulation assays are based on intravenous injection, a complex process that requires circulation through blood, recognition and extravasation through bone marrow vasculature, and migration to a supportive microenvironment 8-11. Thus, some classes of HSCs may remain undetected. By direct intrafemoral injection, we identified rapid SRCs (R-SRCs) within the Lin-CD34 + CD38 lo CD36subpopulation. R-SRCs rapidly generate high levels of human myeloid and erythroid cells within the injected femur, migrate to the blood and colonize individual bones of non-obese diabetic (NOD)-SCID mice within 2 weeks after transplantation. Lentivector-mediated clonal analysis of individual R-SRCs revealed heterogeneity in their proliferative and migratory properties. The identification of a new HSC class and an effective intrafemoral assay provide the tools required to develop more effective stem cell-based therapies that rely on rapid reconstitution.
NATO Science for Peace and Security Series A: Chemistry and Biology, 2008
The hematopoietic system in mammals is comprised of a heterogeneous population of cells which ran... more The hematopoietic system in mammals is comprised of a heterogeneous population of cells which range in function from mature cells of different lineages with limited proliferative potential to multipotent stem cells with extensive proliferative, differentiative and self-renewal capacities. Over a lifetime, the human body produces a trillion blood cells per day. To sustain that enormous cell output, functionally mature cells
Severe combined immunodeficient (SCID) mice transplanted with human bone marrow were treated with... more Severe combined immunodeficient (SCID) mice transplanted with human bone marrow were treated with human mast cell growth factor, a fusion of interleukin-3 and granulocyte-macrophage colony-stimulating factor (PIXY321), or both, starting immediately or 1 month later. Immature human cells repopulated the mouse bone marrow with differentiated human cells of multiple myeloid and lymphoid lineages; inclusion of erythropoietin resulted in human red cells in the peripheral blood. The bone marrow of growth factor-treated mice contained both multipotential and committed myeloid and erythroid progenitors, whereas mice not given growth factors had few human cells and only granulocyte-macrophage progenitors. Thus, this system allows the detection of immature human cells, identification of the growth factors that regulate them, and the establishment of animal models of human hematopoietic diseases.
Hematopoiesis requires tight regulation of the hematopoietic stem cell (HSC) population; however,... more Hematopoiesis requires tight regulation of the hematopoietic stem cell (HSC) population; however, the dynamics of HSC use at steady state are uncertain. Over 3-7 months, we evaluated the repopulation and self-renewal of more than 600 individual human 'severe combined immunodeficiency mouse-repopulating cells' (SRCs), tracked on the basis of lentiviral integration sites, in serially transplanted immune-deficient mice, as well as of SRC daughter cells that migrated to different marrow locations in a single mouse. Our data demonstrate maintenance by self-renewing SRCs after an initial period of clonal instability, a result inconsistent with the clonal succession model. We found wide variation in proliferation kinetics and self-renewal among SRCs, as well as between SRC daughter cells that repopulated equivalently, suggesting that SRC fate is unpredictable before SRCs enter more rigid 'downstream' developmental programs.
In acute myeloid leukemia (AML), the cell of origin, nature and biological consequences of initia... more In acute myeloid leukemia (AML), the cell of origin, nature and biological consequences of initiating lesions and order of subsequent mutations remain poorly understood, as AML is typically diagnosed without observation of a pre-leukemic phase. Here, highly purified hematopoietic stem cells (HSC), progenitor and mature cell fractions from the blood of AML patients were found to contain recurrent DNMT3a mutations (DNMT3a mut) at high allele frequency, but without coincident NPM1 mutations (NPM1c) present in AML blasts. DNMT3a mut-bearing HSC exhibited multilineage repopulation advantage over non-mutated HSC in xenografts, establishing their identity as pre-leukemic-HSC (preL-HSC). preL-HSC were found in remission samples indicating that they survive chemotherapy. Thus DNMT3a mut arises early in AML evolution, likely in HSC, leading to a clonally expanded pool of preL-HSC from which Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:
Objective. The absence of effective strategies for the ex vivo expansion of human hematopoietic s... more Objective. The absence of effective strategies for the ex vivo expansion of human hematopoietic stem cells (HSCs) limits the development of many cell-based therapies. Prior attempts to stimulate HSC expansion have focused on media supplementation using cytokines and growth factors. In these cultures, cellular and microenvironmental compositions change with time. In this study, the impact of controlling these dynamic changes on HSC output is determined. Materials and Methods. Cord blood-derived lin 2 cells were cultured for 8 days in serum-free medium supplemented with stem cell factor, Flt3 ligand, and thrombopoietin. Functional, phenotypic, and molecular (gene and protein) analyses were used to characterize dynamic changes in cellular and microenvironmental composition. The effects of these changes and the mechanism behind their effects on HSC expansion were assessed using a selection/media exchange-based global culture manipulation (GCM) technique. Results. We show that the direct secretion of negative regulators by culture-generated lin 1 cells, and the indirect stimulation of cells to secrete negative regulators by culture-conditioned media, limits in vitro HSC generation. The GCM strategy was able to abrogate these effects to produce elevated numbers of LTC-ICs (14.6-fold relative to input), migrating rapid NOD/ SCID repopulating cells (12.1-fold), and long-term NOD/SCID repopulating cells (5.2-fold). Conclusions. Cellular and microenvironmental changes that occur during all in vitro HSC cultures can significantly affect HSC output through the direct or indirect secretion of negative regulators. This study provides insight into the mechanisms regulating HSC fate in vitro and describes a novel methodology to regulate overall in vitro microenvironmental dynamics to enable the generation of clinically relevant numbers of HSCs.
The nonobese diabetic/severe combined immune deficiency (NOD/SCID) xenotransplantation model has ... more The nonobese diabetic/severe combined immune deficiency (NOD/SCID) xenotransplantation model has emerged as a widely used assay for human hematopoietic stem cells; however, barriers still exist that limit engraftment. We previously identified a short-term SCID-repopulating cell (SRC) following direct intrafemoral injection into NOD/SCID mice, whereas others characterized similar SRCs using NOD/SCID mice depleted of natural killer (NK) cell activity. To determine the model that most efficiently detects short-term SRCs, we compared human engraftment in 6 different xenotransplantation models: NOD/SCID-β2-microglobulin-null mice, anti-CD122 (interleukin-2 receptor β [IL-2Rβ])–treated or unmanipulated NOD/SCID mice, each given transplants by intravenous or intrafemoral injection. Human cell engraftment was highest in intrafemorally injected anti-CD122–treated NOD/SCID mice compared to all other groups at 2 and 6 weeks after transplantation. These modifications to the SRC assay provide im...
Knowledge of the composition and interrelationship of the various hematopoietic stem cells (HSCs)... more Knowledge of the composition and interrelationship of the various hematopoietic stem cells (HSCs) that comprise the human HSC pool and the consequence of culture on each class is required for effective therapies based on stem cells. Clonal tracking of retrovirally transduced HSCs in nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice revealed heterogeneity in the repopulation capacity of SCID-repopulating cells (SRCs). However, it is impossible to establish whether HSC heterogeneity is intrinsic or whether the culture conditions required for retroviral transduction induce qualitative and quantitative alterations to SRCs. Here, we report establishment of a clonal tracking method that uses lentivectors to transduce HSCs with minimal manipulation during overnight culture without cytokine stimulation. By serial bone marrow (BM) sampling of mice receiving transplants, short-term SRCs (ST-SRCs) and long-term SRCs (LT-SRCs) were identified on the basis of repopulation dynamic...
Progress to uncover the molecular and cellular regulators that govern human hematopoietic stem ce... more Progress to uncover the molecular and cellular regulators that govern human hematopoietic stem cell (HSC) fate has been impeded by an inability to obtain highly purified fractions of HSCs. We report that the rhodamine 123 (Rho 123) dye effluxing fraction of the Lin ؊ CD34 ؉ CD38 ؊ population contains SCID-repopulating cells (SRCs) capable of long-term repopulation in primary nono-bese diabetic/severe combined immunodeficient (NOD/SCID) mice. Purification based on Rho uptake led to a 4-fold enrichment of SRCs in the Lin ؊ CD34 ؉ CD38 ؊ fraction, with a frequency of 1 SRC in 30 Lin ؊ CD34 ؉ CD38 ؊ Rho lo cells. The Lin ؊ CD34 ؉ CD38 ؊ Rho lo fraction also possesses long-term selfrenewal capacity as measured by serial transplantation totaling more than 20 weeks. We conclude that Rho dye efflux provides an additional means of purifying human HSCs in the quest to achieve homogeneous populations of primitive cells for both experimental and therapeutic applications. (Blood.
Ł € Hematopoiesis and Stem Cells (3132 articles)Ł € Gene Therapy (524 articles)Articles on simila... more Ł € Hematopoiesis and Stem Cells (3132 articles)Ł € Gene Therapy (524 articles)Articles on similar topics can be found in the following Blood collectionshttp://bloodjournal.hematologylibrary.org/site/misc/rights.xhtml#repub_requests Information about reproducing this article in parts or in its entirety may be found online at:http://bloodjournal.hematologylibrary.org/site/misc/rights.xhtml#reprints Information about ordering reprints may be found online at:http://bloodjournal.hematologylibrary.org/site/subscriptions/index.xhtml Information about subscriptions and ASH membership may be found online at:
Leukemia in humans arises from the multistep accumulation of mutations. However, the identity of ... more Leukemia in humans arises from the multistep accumulation of mutations. However, the identity of the cell of origin, the nature of the first genetic lesion and the order of subsequent mutations remain poorly understood, as most cases of de novo acute myeloid leukemia (AML) are diagnosed without prior observation of a pre-leukemic clonal expansion. As part of studies to examine intra-tumoral genetic heterogeneity in AML, we carried out deep targeted sequencing (read depth 250×) of 101 commonly mutated leukemia genes on samples from 12 patients at diagnosis. Normal T-cells from each sample were expanded in vitro to provide a non-leukemic hematopoietic tissue for comparison. In 3 of 4 patients, we unexpectedly identified DNMT3a mutation not only in AML cells but also in T-cells at a low allele frequency (1-20%). Other genetic alterations such as NPM1 mutation (mutNPM1) were found only in AML cells and not in T-cells, ruling out contaminating AML cells as the source of the DNMT3a signal...
In acute myeloid leukaemia, long-term survival is poor as most patients relapse despite achieving... more In acute myeloid leukaemia, long-term survival is poor as most patients relapse despite achieving remission. Historically, the failure of therapy has been thought to be due to mutations that produce drug resistance, possibly arising as a consequence of the mutagenic properties of chemotherapy drugs. However, other lines of evidence have pointed to the pre-existence of drug-resistant cells. For example, deep sequencing of paired diagnosis and relapse acute myeloid leukaemia samples has provided direct evidence that relapse in some cases is generated from minor genetic subclones present at diagnosis that survive chemotherapy, suggesting that resistant cells are generated by evolutionary processes before treatment and are selected by therapy. Nevertheless, the mechanisms of therapy failure and capacity for leukaemic regeneration remain obscure, as sequence analysis alone does not provide insight into the cell types that are fated to drive relapse. Although leukaemia stem cells have been linked to relapse owing to their dormancy and self-renewal properties, and leukaemia stem cell gene expression signatures are highly predictive of therapy failure, experimental studies have been primarily correlative and a role for leukaemia stem cells in acute myeloid leukaemia relapse has not been directly proved. Here, through combined genetic and functional analysis of purified subpopulations and xenografts from paired diagnosis/relapse samples, we identify therapy-resistant cells already present at diagnosis and two major patterns of relapse. In some cases, relapse originated from rare leukaemia stem cells with a haematopoietic stem/progenitor cell phenotype, while in other instances relapse developed from larger subclones of immunophenotypically committed leukaemia cells that retained strong stemness transcriptional signatures. The identification of distinct patterns of relapse should lead to improved methods for disease management and monitoring in acute myeloid leukaemia. Moreover, the shared functional and transcriptional stemness properties that underlie both cellular origins of relapse emphasize the importance of developing new therapeutic approaches that target stemness to prevent relapse.
Based on initial observations of human CD34+ Thy-1+ cells and long-term culture-initiating cells ... more Based on initial observations of human CD34+ Thy-1+ cells and long-term culture-initiating cells (LTC-IC) in the bone marrow of some sublethally irradiated severe combined immunodeficient (SCID) mice transplanted intravenously with normal human marrow cells, and the subsequent finding that the NOD/LtSz-scid/scid (NOD/SCID) mouse supports higher levels of human cell engraftment, we undertook a series of time course experiments to examine posttransplant changes in the number, tissue distribution, cycling activity, and in vivo differentiation pattern of various human hematopoietic progenitor cell populations in this latter mouse model. These studies showed typical rapid posttransplant recovery curves for human CD34- CD19+ (B-lineage) cells, CD34+ granulopoietic, erythroid, and multilineage colony-forming cells (CFC), LTC-IC, and CD34+ Thy-1+ cells from a small initial population representing <0.1% of the original transplant. The most primitive human cell populations reached maximum ...
The ability to transfer new genetic material into human hematopoietic cells provides the foundati... more The ability to transfer new genetic material into human hematopoietic cells provides the foundation for characterizing the organization and developmental program of human hematopoietic stem cells. It also provides a valuable model in which to test gene transfer and long-term expression in human hematopoietic cells as a prelude to human gene therapy. At the present time such studies are limited by the absence of in vivo assays for human stem cells, although recent descriptions of the engraftment of human hematopoietic cells in immune-deficient mice may provide the basis for such an assay. This study focuses on the establishment of conditions required for high efficiency retrovirus-mediated gene transfer into human hematopoietic progenitors that can be assayed in vitro in short-term colony assays and in vivo in immune-deficient mice. Here we report that a 24-hour preincubation of human bone marrow in 5637-conditioned medium, before infection, increases gene transfer efficiency into in...
Fanconi anemia (FA) is a pleiotropic inherited disease that causes bone marrow failure in childre... more Fanconi anemia (FA) is a pleiotropic inherited disease that causes bone marrow failure in children. However, the specific involvement of FA genes in hematopoiesis and their relation to bone marrow (BM) failure is still unclear. The increased sensitivity of FA cells to DNA cross-linking agents such as mitomycin C (MMC) and diepoxybutane (DEB), including the induction of chromosomal aberrations and delay in the G2 phase of the cell cycle, have suggested a role for the FA genes in DNA repair, cell cycle regulation, and apoptosis. We previously reported the cloning of the FA group C gene (FAC) and the generation of a Fac mouse model. Surprisingly, the Fac -/- mice did not show any of the hematologic defects found in FA patients. To better understand the relationship of FA gene functions to BM failure, we have analyzed the in vivo effect of an FA-specific DNA damaging agent in Fac -/- mice. The mice were found to be highly sensitive to DNA cross-linking agents; acute exposure to MMC prod...
We have previously reported the development of in vivo functional assays for primitive human hema... more We have previously reported the development of in vivo functional assays for primitive human hematopoietic cells based on their ability to repopulate the bone marrow (BM) of severe combined immunodeficient (SCID) and nonobese diabetic/SCID (NOD/SCID) mice following intravenous transplantation. Accumulated data from gene marking and cell purification experiments indicate that the engrafting cells (defined as SCID-repopulating cells or SRC) are biologically distinct from and more primitive than most cells that can be assayed in vitro. Here we demonstrate through limiting dilution analysis that the NOD/SCID xenotransplant model provides a quantitative assay for SRC. Using this assay, the frequency of SRC in cord blood (CB) was found to be 1 in 9.3 x 10(5) cells. This was significantly higher than the frequency of 1 SRC in 3.0 x 10(6) adult BM cells or 1 in 6.0 x 10(6) mobilized peripheral blood (PB) cells from normal donors. Mice transplanted with limiting numbers of SRC were engrafted...
A major problem hampering effective stem cell-based therapies is the absence of a clear understan... more A major problem hampering effective stem cell-based therapies is the absence of a clear understanding of the human hematopoietic stem cell (HSC) pool composition. The severe combined immunodeficiency (SCID) repopulating cell (SRC) xenotransplant assay system provides a powerful tool for characterizing the frequency, cell surface markers, cell cycle status, homing and response to cytokine stimulation of human HSCs 1-3. Clonal tracking of retrovirally transduced SRCs and transplantation of specific subpopulations revealed SRC classes with distinct repopulation potentials 4-7. However, all HSC repopulation assays are based on intravenous injection, a complex process that requires circulation through blood, recognition and extravasation through bone marrow vasculature, and migration to a supportive microenvironment 8-11. Thus, some classes of HSCs may remain undetected. By direct intrafemoral injection, we identified rapid SRCs (R-SRCs) within the Lin-CD34 + CD38 lo CD36subpopulation. R-SRCs rapidly generate high levels of human myeloid and erythroid cells within the injected femur, migrate to the blood and colonize individual bones of non-obese diabetic (NOD)-SCID mice within 2 weeks after transplantation. Lentivector-mediated clonal analysis of individual R-SRCs revealed heterogeneity in their proliferative and migratory properties. The identification of a new HSC class and an effective intrafemoral assay provide the tools required to develop more effective stem cell-based therapies that rely on rapid reconstitution.
NATO Science for Peace and Security Series A: Chemistry and Biology, 2008
The hematopoietic system in mammals is comprised of a heterogeneous population of cells which ran... more The hematopoietic system in mammals is comprised of a heterogeneous population of cells which range in function from mature cells of different lineages with limited proliferative potential to multipotent stem cells with extensive proliferative, differentiative and self-renewal capacities. Over a lifetime, the human body produces a trillion blood cells per day. To sustain that enormous cell output, functionally mature cells
Severe combined immunodeficient (SCID) mice transplanted with human bone marrow were treated with... more Severe combined immunodeficient (SCID) mice transplanted with human bone marrow were treated with human mast cell growth factor, a fusion of interleukin-3 and granulocyte-macrophage colony-stimulating factor (PIXY321), or both, starting immediately or 1 month later. Immature human cells repopulated the mouse bone marrow with differentiated human cells of multiple myeloid and lymphoid lineages; inclusion of erythropoietin resulted in human red cells in the peripheral blood. The bone marrow of growth factor-treated mice contained both multipotential and committed myeloid and erythroid progenitors, whereas mice not given growth factors had few human cells and only granulocyte-macrophage progenitors. Thus, this system allows the detection of immature human cells, identification of the growth factors that regulate them, and the establishment of animal models of human hematopoietic diseases.
Hematopoiesis requires tight regulation of the hematopoietic stem cell (HSC) population; however,... more Hematopoiesis requires tight regulation of the hematopoietic stem cell (HSC) population; however, the dynamics of HSC use at steady state are uncertain. Over 3-7 months, we evaluated the repopulation and self-renewal of more than 600 individual human 'severe combined immunodeficiency mouse-repopulating cells' (SRCs), tracked on the basis of lentiviral integration sites, in serially transplanted immune-deficient mice, as well as of SRC daughter cells that migrated to different marrow locations in a single mouse. Our data demonstrate maintenance by self-renewing SRCs after an initial period of clonal instability, a result inconsistent with the clonal succession model. We found wide variation in proliferation kinetics and self-renewal among SRCs, as well as between SRC daughter cells that repopulated equivalently, suggesting that SRC fate is unpredictable before SRCs enter more rigid 'downstream' developmental programs.
In acute myeloid leukemia (AML), the cell of origin, nature and biological consequences of initia... more In acute myeloid leukemia (AML), the cell of origin, nature and biological consequences of initiating lesions and order of subsequent mutations remain poorly understood, as AML is typically diagnosed without observation of a pre-leukemic phase. Here, highly purified hematopoietic stem cells (HSC), progenitor and mature cell fractions from the blood of AML patients were found to contain recurrent DNMT3a mutations (DNMT3a mut) at high allele frequency, but without coincident NPM1 mutations (NPM1c) present in AML blasts. DNMT3a mut-bearing HSC exhibited multilineage repopulation advantage over non-mutated HSC in xenografts, establishing their identity as pre-leukemic-HSC (preL-HSC). preL-HSC were found in remission samples indicating that they survive chemotherapy. Thus DNMT3a mut arises early in AML evolution, likely in HSC, leading to a clonally expanded pool of preL-HSC from which Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:
Objective. The absence of effective strategies for the ex vivo expansion of human hematopoietic s... more Objective. The absence of effective strategies for the ex vivo expansion of human hematopoietic stem cells (HSCs) limits the development of many cell-based therapies. Prior attempts to stimulate HSC expansion have focused on media supplementation using cytokines and growth factors. In these cultures, cellular and microenvironmental compositions change with time. In this study, the impact of controlling these dynamic changes on HSC output is determined. Materials and Methods. Cord blood-derived lin 2 cells were cultured for 8 days in serum-free medium supplemented with stem cell factor, Flt3 ligand, and thrombopoietin. Functional, phenotypic, and molecular (gene and protein) analyses were used to characterize dynamic changes in cellular and microenvironmental composition. The effects of these changes and the mechanism behind their effects on HSC expansion were assessed using a selection/media exchange-based global culture manipulation (GCM) technique. Results. We show that the direct secretion of negative regulators by culture-generated lin 1 cells, and the indirect stimulation of cells to secrete negative regulators by culture-conditioned media, limits in vitro HSC generation. The GCM strategy was able to abrogate these effects to produce elevated numbers of LTC-ICs (14.6-fold relative to input), migrating rapid NOD/ SCID repopulating cells (12.1-fold), and long-term NOD/SCID repopulating cells (5.2-fold). Conclusions. Cellular and microenvironmental changes that occur during all in vitro HSC cultures can significantly affect HSC output through the direct or indirect secretion of negative regulators. This study provides insight into the mechanisms regulating HSC fate in vitro and describes a novel methodology to regulate overall in vitro microenvironmental dynamics to enable the generation of clinically relevant numbers of HSCs.
The nonobese diabetic/severe combined immune deficiency (NOD/SCID) xenotransplantation model has ... more The nonobese diabetic/severe combined immune deficiency (NOD/SCID) xenotransplantation model has emerged as a widely used assay for human hematopoietic stem cells; however, barriers still exist that limit engraftment. We previously identified a short-term SCID-repopulating cell (SRC) following direct intrafemoral injection into NOD/SCID mice, whereas others characterized similar SRCs using NOD/SCID mice depleted of natural killer (NK) cell activity. To determine the model that most efficiently detects short-term SRCs, we compared human engraftment in 6 different xenotransplantation models: NOD/SCID-β2-microglobulin-null mice, anti-CD122 (interleukin-2 receptor β [IL-2Rβ])–treated or unmanipulated NOD/SCID mice, each given transplants by intravenous or intrafemoral injection. Human cell engraftment was highest in intrafemorally injected anti-CD122–treated NOD/SCID mice compared to all other groups at 2 and 6 weeks after transplantation. These modifications to the SRC assay provide im...
Knowledge of the composition and interrelationship of the various hematopoietic stem cells (HSCs)... more Knowledge of the composition and interrelationship of the various hematopoietic stem cells (HSCs) that comprise the human HSC pool and the consequence of culture on each class is required for effective therapies based on stem cells. Clonal tracking of retrovirally transduced HSCs in nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice revealed heterogeneity in the repopulation capacity of SCID-repopulating cells (SRCs). However, it is impossible to establish whether HSC heterogeneity is intrinsic or whether the culture conditions required for retroviral transduction induce qualitative and quantitative alterations to SRCs. Here, we report establishment of a clonal tracking method that uses lentivectors to transduce HSCs with minimal manipulation during overnight culture without cytokine stimulation. By serial bone marrow (BM) sampling of mice receiving transplants, short-term SRCs (ST-SRCs) and long-term SRCs (LT-SRCs) were identified on the basis of repopulation dynamic...
Uploads
Papers by Monica Doedens