Biomedical subjects

Monica Doedens

Publications and source records attributed to Monica Doedens.

7 recordsLinked to original sources

PubMed2006

Individual stem cells with highly variable proliferation and self-renewal properties comprise the human hematopoietic stem cell compartment.

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.

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PubMed2006

Low rhodamine 123 retention identifies long-term human hematopoietic stem cells within the Lin-CD34+CD38- population.

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 nonobese 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-Rholo cells. The Lin-CD34+CD38-Rholo fraction also possesses long-term self-renewal 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.

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PubMed2005

Human short-term repopulating stem cells are efficiently detected following intrafemoral transplantation into NOD/SCID recipients depleted of CD122+ cells.

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-beta2-microglobulin-null mice, anti-CD122 (interleukin-2 receptor beta [IL-2Rbeta])-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 improved detection of human stem cells and demonstrate that CD122+ cells provide barriers to stem cell engraftment, a finding with potential clinical relevance.

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PubMed2005

Dynamic changes in cellular and microenvironmental composition can be controlled to elicit in vitro human hematopoietic stem cell expansion.

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(-) 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(+) 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.

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PubMed2003

Lentivector-mediated clonal tracking reveals intrinsic heterogeneity in the human hematopoietic stem cell compartment and culture-induced stem cell impairment.

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 dynamics demonstrating that their existence is not an experimental artifact but reflects the state of the HSC pool. However, 4 days of culture in conditions previously used for SRC retroviral transduction significantly reduced SRC number as assessed by clonal analysis. These studies provide a foundation to understand the molecular and cellular determinants of human HSC development and to develop therapies targeted to specific HSC classes.

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PubMed2003

Rapid myeloerythroid repopulation after intrafemoral transplantation of NOD-SCID mice reveals a new class of human stem cells.

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. Clonal tracking of retrovirally transduced SRCs and transplantation of specific subpopulations revealed SRC classes with distinct repopulation potentials. 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. Thus, some classes of HSCs may remain undetected. By direct intrafemoral injection, we identified rapid SRCs (R-SRCs) within the Lin-CD34+CD38loCD36- subpopulation. 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.

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PubMed2003

Characterization of cord blood hematopoietic stem cells.

A major problem hampering the development of effective stem cell-based therapies is the absence of a clear understanding of the composition of the hematopoietic stem cell (HSC) pool in humans and how ex vivo manipulation can differentially affect the various HSC classes. This paper will review recent advances in the use of the NOD/SCID xenotransplant assay to characterize the human stem cell compartment and to determine how ex vivo culture affects stem cells. Using lentivector-mediated clonal tracking we found that only 4 days of culture can significantly reduce the number of SCID-repopulating cells (SRCs) contributing to the human graft. Similar results were seen with a competitive assay strategy where non-cultured cells marked with the RFP-lentivector markedly outcompete cultured cells marked with a EGFP-lentivector both transplanted into the same NOD/SCID mouse. A novel intrafemoral (IF) assay was developed to permit the transplantation of human stem cells that might be difficult to detect using the traditional IV injection method. With the IF assay we identified a novel class of human stem cell with the ability to rapidly generate a large graft of human myeloid and erythroid cells within 2 weeks post transplant.

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