Ability Of Hematopoietic Stem Cells Research Articles

Retrovirus-mediated gene transfer to human hematopoietic stem cells.

The ability of hematopoietic stem cells (HSCs) to engraft in a recipient and establish long-term repopulation of the hematopoietic system makes them ideal targets for gene therapy vectors designed to correct inherited or acquired diseases affecting the hematopoietic and immune systems.

Negative Regulation by Interleukin-3 (IL-3) of Mouse Early B-Cell Progenitors and Stem Cells in Culture: Transduction of the Negative Signals by βc and βIL-3 Proteins of IL-3 Receptor and Absence of Negative Regulation by Granulocyte-Macrophage Colony-Stimulating Factor

AbstractThe receptors for interleukin-3 (IL-3), granulocyte-macrophage colony-stimulating factor (GM-CSF), and IL-5 share a common signaling subunit βc. However, in the mouse, there is an additional IL-3 signaling protein, βIL-3, which is specific for IL-3. We have previously reported that IL-3 abrogates the lymphoid potentials of murine lymphohematopoietic progenitors and the reconstituting ability of hematopoietic stem cells. We used bone marrow cells from βc- and βIL-3–knock-out mice to examine the relative contributions of the receptor proteins to the negative regulation by IL-3. First, we tested the effects of IL-3 on lymphohematopoietic progenitors by using lineage-negative (Lin−) marrow cells of 5-fluorouracil (5-FU)-treated mice in the two-step methylcellulose culture we reported previously. Addition of IL-3 to the combination of steel factor (SF, c-kit ligand) and IL-11 abrogated the B-lymphoid potential of the marrow cells of both types of knock-out mice as well as wild-type mice. Next, we investigated the effects of IL-3 on in vitro expansion of the hematopoietic stem cells. We cultured Lin−Sca-1–positive, c-kit–positive marrow cells from 5-FU–treated mice in suspension in the presence of SF and IL-11 with or without IL-3 for 7 days and tested the reconstituting ability of the cultured cells by transplanting the cells into lethally irradiated Ly-5 congenic mice together with “compromised” marrow cells. Presence of IL-3 in culture abrogated the reconstituting ability of the cells from both types of knock-out mice and the wild-type mice. In contrast, addition of GM-CSF to the suspension culture abrogated neither B-cell potential nor reconstituting abilities of the cultured cells of wild-type mice. These observations may have implications in the choice of cytokines for use in in vitro expansion of human hematopoietic stem cells and progenitors.© 1998 by The American Society of Hematology.

Negative Regulation by Interleukin-3 (IL-3) of Mouse Early B-Cell Progenitors and Stem Cells in Culture: Transduction of the Negative Signals by βc and βIL-3 Proteins of IL-3 Receptor and Absence of Negative Regulation by Granulocyte-Macrophage Colony-Stimulating Factor

The receptors for interleukin-3 (IL-3), granulocyte-macrophage colony-stimulating factor (GM-CSF), and IL-5 share a common signaling subunit βc. However, in the mouse, there is an additional IL-3 signaling protein, βIL-3, which is specific for IL-3. We have previously reported that IL-3 abrogates the lymphoid potentials of murine lymphohematopoietic progenitors and the reconstituting ability of hematopoietic stem cells. We used bone marrow cells from βc- and βIL-3–knock-out mice to examine the relative contributions of the receptor proteins to the negative regulation by IL-3. First, we tested the effects of IL-3 on lymphohematopoietic progenitors by using lineage-negative (Lin−) marrow cells of 5-fluorouracil (5-FU)-treated mice in the two-step methylcellulose culture we reported previously. Addition of IL-3 to the combination of steel factor (SF, c-kit ligand) and IL-11 abrogated the B-lymphoid potential of the marrow cells of both types of knock-out mice as well as wild-type mice. Next, we investigated the effects of IL-3 on in vitro expansion of the hematopoietic stem cells. We cultured Lin−Sca-1–positive, c-kit–positive marrow cells from 5-FU–treated mice in suspension in the presence of SF and IL-11 with or without IL-3 for 7 days and tested the reconstituting ability of the cultured cells by transplanting the cells into lethally irradiated Ly-5 congenic mice together with “compromised” marrow cells. Presence of IL-3 in culture abrogated the reconstituting ability of the cells from both types of knock-out mice and the wild-type mice. In contrast, addition of GM-CSF to the suspension culture abrogated neither B-cell potential nor reconstituting abilities of the cultured cells of wild-type mice. These observations may have implications in the choice of cytokines for use in in vitro expansion of human hematopoietic stem cells and progenitors. © 1998 by The American Society of Hematology.

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

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

Gene Transfer into Murine Hematopoietic Stem Cells and Bone Marrow Stromal Cells

The use of recombinant retroviral vectors to transfer genetic sequences into hematopoietic stem cells (HSC) is one approach to somatic gene therapy. Two limitations of such retroviral vectors are the degree of efficiency of transfer into the reconstituting hematopoietic stem cells and the loss of reconstituting ability of hematopoietic stem cells when manipulated in vitro during infection and selection. We have investigated the effects on the efficiency of gene transfer of prestimulation of hematopoietic stem cells by growth factors prior to infection. Prestimulation of bone marrow cells in WEHI-3b-conditioned media improved the efficiency of gene transfer into CFU-S stem cells. The majority of animals transplanted with bone marrow infected after prestimulation with a simplified retrovirus, Zip PGK ADA, demonstrated long-term and stable expression of human adenosine deaminase (ADA) after full hematopoietic reconstitution. In separate experiments, retroviral vectors have been used to transfer the SV40 large T antigen sequences into stromal cells making up the hematopoietic microenvironment. Stromal cells expressing large T antigen are immortalized, and some support the maintenance of day 12 CFU-S (CFU-S12) and reconstituting hematopoietic stem cells in vitro for up to 4 weeks. Such immortalized stromal cell lines provide an in vitro hematopoietic microenvironment which may allow prolonged in vitro manipulations during infection and selection of hematopoietic stem cells without loss of reconstituting ability. We are using immortalized stromal cell lines resistant to deoxycoformycin (dCF) to select transduced murine HSC containing human ADA in vitro. The use of recombinant retroviral vectors provides a promising approach to correction of human diseases involving bone marrow cells.

AGING AND HEMATOPOIESIS

The proliferative ability of hematopoietic stem cells (HSC) was compared in young and aged mice. Infusion of coisogeneic marrow cures the hematopoietic defect of the W/Wv, a mouse with a recessively inherited defect in stem cells, including HSC. W/Wv were cured with equal frequency by relatively small doses of marrow (5 X 10(4) nucleated cells, an average of 2-3 "curative" HSC per aliquot) from 3-month-old or 27-month-old +/+ donors. After functioning in the originally cured W/Wv for 26 months, the marrow was used to cure other W/Wv. After functioning in secondary recipients for 16 months, it was, in turn, used to cure still other W/Wv. There was no difference between "old" and "young" bone marrow with respect to the frequency of cure in W/Wv, the duration of cure--or, in regard to marrow from cured W/Wv, the number of nucleated and peroxidase-positive cells per humerus and the number of cells capable of producing spleen colonies in irradiated recipients. Thus, these studies fail to disclose any evidence for a proliferative limitation for old as compared to young HSC.

Effect of the thymus on the ability of hematopoietic stem cells to recover after sublethal radiation injury

Following fractional irradiation of the mouse bone marrow (300 rad in vitro + 300 rad in vivo) the damage of stem hemopoietic cells was more pronounced when the interval between the irradiations was 30 min as compared to five hrs, on account of recovery from sublethal radiation injury (Elkind's repair of radiation damages). In adult thymectomized mice the capability of stem hemopoietic cells to Elkind's repair was sharply deranged 2.5 to 5 months after the operation. This effect of thymectomy was abrogated by the newborn thymus graft.