Fraction Of Hematopoietic Stem Cells Research Articles

Identification of Novel Regulators of Hematopoietic Stem Cell Mobilization.

In an effort to identify the molecular changes during hematopoietic stem cell (HSC) mobilization we have analyzed genome wide changes in gene expression in HSCs in response to the chemotherapeutic agent cyclophosphamide (Cy) and the growth factor granulocyte colony stimulating factor (G). Cy/G treatment leads to an initial loss of proliferating precursors, followed by a rapid expansion of stem and progenitor cells and their migration to the peripheral blood. While this approach has been capitalized on for harvesting hematopoietic stem cells in clinical therapy, the molecular regulation of this process remains less well understood. To analyze changes in gene expression that occur in vivo as HSCs proliferate and prepare to migrate out of the bone marrow, we enriched for HSC fractions by isolating cells that express c-kit (K), Sca-1(S) and low levels of Thy1.1(T) but do not express lineage (L) markers (KTLS) from untreated mice and mice treated with Cy, G or Cy/G. The gene expression profile of these cells was examined by microarray analysis, relative to HSCs isolated from untreated mice. Interestingly, among the genes upregulated by Cy/G, a large majority (>60%) were a consequence of synergistic activity between Cy and G, while the rest of the upregulated genes could be attributed to activation by Cy or G alone. To test whether this screen allowed identification of novel candidates that regulate HSC function we analyzed the role of transforming growth factor beta induced gene (Big-h3), a gene highly upregulated after Cy/G treatment. Big-h3 is an extracellular matrix protein that mediates the adhesion and migration of various cell types, however its role in HSCs is unknown. Our experiments indicate that overexpression of Big-h3 in HSCs leads to enhanced adhesion to fibronectin as well as upregulation of specific integrins. These experiments suggest that changes in adhesion are an integral component of HSC mobilization, and that Big-h3 is one regulator of such processes. Further functional analysis of other candidates will likely lead to identification of other novel regulators of HSC development and mobilization.

Beta-galactosidase of ROSA26 mice is a useful marker for detecting the definitive erythropoiesis after stem cell transplantation.

Hematopoietic reconstitution after stem cell transplantation has been analyzed by using stem cells of Ly5 congenic mice. However, the early erythropoiesis has never been analyzed because this marker is not expressed on all of the erythroid lineage cells. The transgenic mouse expressing beta-galactosidase (beta-gal) or green fluorescent protein (GFP) has been reported. Using these markers, we analyzed the early erythropoiesis after stem cell transplantation. The beta-gal activity and GFP were examined in the hematopoietic cells of ROSA26 and GFP transgenic mice, respectively, by flow cytometry. The primitive hematopoietic stem cell fraction (Lin(-)c-kit(+)Sca-1(+)) in bone marrow (BM) cells of ROSA26 mice was transferred into lethally irradiated mice. The kinetics of hematopoietic reconstitution was analyzed in the BM and spleen after transplantation. The beta-gal activity, but not the GFP and Ly5, was detected in all of the erythroid (TER119+) cells. The beta-gal activity was also detected in the donor-derived myeloid (Mac-1+), B lymphoid (B220+), and T lymphoid (Thy-1+) cells in the BM and spleen after stem cell transplantation. The kinetics of the hematopoietic reconstitution demonstrated that early erythroid (TER119(low)CD71(med)) cells were developed in the BM and spleen within 2 days after transplantation before development of proerythroblasts (TER119(+)CD71(high)), and that massive erythropoiesis and myelopoiesis were observed in the spleen until 2 and 4 weeks after transplantation, respectively. Conclusions. The beta-gal of ROSA26 mice can be a useful marker to identify the donor-derived hematopoietic cells, including early erythroid cells, and the first major wave of erythropoiesis occurring in the spleen after stem cell transplantation.

Molecular cloning, sequence and expression pattern analysis of the mouse orthologue of the leukemia-associated guanine nucleotide exchange factor

Guanine nucleotide exchange factors (GEFs) of the Dbl family activate Rho proteins and thereby participate in diverse signaling pathways involving this family of small GTPases. However, specific role of Dbl-GEFs in developmental processes, particularly cell differentiation, remains largely unexplored. Recently, a novel member of the Dbl family, leukemia-associated Rho GEF factor (LARG), has been reported to be a fusion partner with mixed-lineage leukemia (MLL) protein in the acute myeloid leukemia, suggesting potential involvement of LARG in regulation of hematopoiesis. In this study, we describe the isolation of the cDNA copy and analysis of genomic structure and expression profile of the mouse orthologue of the human LARG gene. The mouse LARG ( mLARG) gene contains 42 exons. The mLARG cDNA is 10,040 bp long and contains a 4631-bp open reading frame (ORF). The predicted mLARG protein shares an overall 89% identity with its human counterpart and contains the same functional domains, namely, Dbl homology (DH), pleckstrin homology (PH), PSD-95, Dlg and ZO-1/2 (PDZ) and regulator of G protein signaling (RGS) domains, as well as two putative signals of nuclear localization. The mLARG message is expressed in all tissues studied, with comparably higher expression levels observed in brain, lung, testis, liver and heart. Using amplified cDNA samples from sorted hematopoietic cells, we showed that mLARG is highly expressed in hematopoietic stem cell fractions, as well as in immature erythroid cells. These results demonstrate high similarity between mouse and human LARG proteins and genes and provide further support to the hypothesis of the potential involvement of LARG in regulation of early stages of hematopoiesis.

Gene expression profile in bone marrow and hematopoietic stem cells in mice exposed to inhaled benzene

Acute myeloid leukemia and chronic lymphocytic leukemia are associated with benzene exposure. In mice, benzene induces chromosomal breaks as a primary mode of genotoxicity in the bone marrow (BM). Benzene-induced DNA lesions can lead to changes in hematopoietic stem cells (HSC) that give rise to leukemic clones. To gain insight into the mechanism of benzene-induced leukemia, we investigated the DNA damage repair and response pathways in total bone marrow and bone marrow fractions enriched for HSC from male 129/SvJ mice exposed to benzene by inhalation. Mice exposed to 100 ppm benzene for 6 h per day, 5 days per week for 2 week showed significant hematotoxicity and genotoxicity compared to air-exposed control mice. Benzene exposure did not alter the level of apoptosis in BM or the percentage of HSC in BM. RNA isolated from total BM cells and the enriched HSC fractions from benzene-exposed and air-exposed mice was used for microarray analysis and quantitative real-time RT-PCR. Interestingly, mRNA levels of DNA repair genes representing distinct repair pathways were largely unaffected by benzene exposure, whereas altered mRNA expression of various apoptosis, cell cycle, and growth control genes was observed in samples from benzene-exposed mice. Differences in gene expression profiles were observed between total BM and HSC. Notably, p21 mRNA was highly induced in BM but was not altered in HSC following benzene exposure. The gene expression pattern suggests that HSC isolated immediately following a 2 weeks exposure to 100 ppm benzene were not actively proliferating. Understanding the toxicogenomic profile of the specific target cell population involved in the development of benzene-associated diseases may lead to a better understanding of the mechanism of benzene-induced leukemia and may identify important interindividual and tissue susceptibility factors.

Hematopoietic Stem Cells Expressing the Myeloid Lysozyme Gene Retain Long-Term, Multilineage Repopulation Potential

Single cell PCR studies showed that hematopoietic stem cells (HSCs) express a variety of lineage-affiliated genes. However, it remains unclear whether these cells exhibiting “lineage priming” represent bona fide stem cells or a subpopulation earmarked for differentiation. Here we have used a Cre-Lox approach to follow the fate of cells expressing a lineage-affiliated marker. We crossed lysozyme Cre mice with yellow fluorescent protein (EYFP) reporter mice and found EYFP gene expression not only in myelomonocytic cells but also in a fraction of HSCs as well as B cells and T cells. Transplantation of EYFP + HSCs into primary and secondary recipients generated mice in which all hematopoietic cells were EYFP +. In contrast, crosses between CD19 Cre and lck Cre mice with reporter mice showed no EYFP expression in HSCs or intermediate progenitors. Our results demonstrate that lysozyme expression does not mark myeloid commitment and that long-term repopulation potential is maintained in primed HSCs.

Hematopoietic stem cells engraft in mice with absolute efficiency.

The engraftment of murine hematopoietic stem cells (HSCs) into irradiated mice is thought to be an inefficient process, but has yet to be measured directly. We used two independent strategies to test their engraftment efficiency: one measured competition of unpurified donor bone marrow cells with recipient cells in murine hosts and the other tracked the engraftment of one highly purified stem cell injected per recipient. The results showed that stem cells engrafted with near absolute efficiency. Thus, inefficient engraftment cannot explain the low frequency of permanent reconstitutions observed with pure HSC fractions and instead suggests most initially engrafted cells fail to sustain self-renewal.

Nutrition status and radiation-induced cancer in mice

Dietary restriction, especially caloric restriction (CR), is a major carcinogenic modifier during experimental carcinogenesis. We attempted to examine the effects of CR on radiation-induced myeloid leukemia (MyL). The spontaneous incidence of MyL in C3H/He mice is 1.1%, and the incidence increases to 21.6% by X-ray irradiation. However, the incidence was decreased in the CR groups; it was 7.9% in RA (life-span restricted diet from the age of 6 weeks), 10.7% in RB (restricted diet after irradiation), 16.2% in RC (restricted from 6 weeks old until irradiation at 10 weeks old). The differences between both the RA and RB groups and the non-restricted group were statistically significant. The significantly fewer hematopoietic stem cells (HSC), potential target cells for radiation leukemia in the RA and RC at the time of radiation exposure, and the smaller cycling fraction of HSC under CR in the femur of restricted mice (26±4.5%) than in the non-restricted group (44±20%), were responsible for this underlying mechanism. CR contributes to the incidence reduction of leukemia at the initiation stage of leukemogenesis and, more significantly, to the reduction of MyL during the promotion stage of radiation leukemogenesis.

STAT5 promotes multilineage hematolymphoid development in vivo through effects on early hematopoietic progenitor cells

The transcription factor signal transducers and activators of transcription 5 (STAT5) is activated by numerous cytokines that orchestrate blood cell development. Multilineage peripheral blood cytopenias were observed in adult mice lacking both isoforms of STAT5 (STAT5A and STAT5B) as well as accelerated rates of apoptosis in the bone marrow. Although the hematopoietic stem cell (HSC) population was preserved in a number of these mice, the post-HSC progenitor populations were diminished and a marked reduction in functional progenitors (spleen colony-forming units) was detected. Competitive bone marrow transplantation studies in vivo revealed a profound impairment of repopulation potential of STAT5-null HSCs, leading to complete lack of contribution to the myeloid, erythroid, and lymphoid lineages. These abnormalities were associated with heightened proliferation activity in the HSC fraction, suggesting the action of homeostatic mechanisms to maintain sufficient levels of diverse blood cell types for viability. Thus, STAT5 normally sustains the robust hematopoietic reserve that contributes to host viability through crucial survival effects on early progenitor cells.

Identification of myelodysplastic syndrome–specific genes by DNA microarray analysis with purified hematopoietic stem cell fraction

Myelodysplastic syndrome (MDS) is a slowly progressing hematologic malignancy associated with a poor outcome. Despite the relatively high incidence of MDS in the elderly, differentiation of MDS from de novo acute myeloid leukemia (AML) still remains problematic. Identification of genes expressed in an MDS-specific manner would allow the molecular diagnosis of MDS. Toward this goal, AC133 surface marker-positive hematopoietic stem cell (HSC)-like fractions have been collected from a variety of leukemias in a large-scale and long-term genomics project, referred to as "Blast Bank," and transcriptome of these purified blasts from the patients with MDS were then compared with those from AML through the use of oligonucleotide microarrays. A number of genes were shown to be expressed in a disease-specific manner either to MDS or AML. Among the former found was the gene encoding the protein Delta-like (Dlk) that is distantly related to the Delta-Notch family of signaling proteins. Because overexpression of Dlk may play a role in the pathogenesis of MDS, the disease specificity of Dlk expression was tested by a quantitative "real-time" polymerase chain reaction analysis. Examination of the Blast Bank samples from 22 patients with MDS, 31 with AML, and 8 with chronic myeloid leukemia confirmed the highly selective expression of the Dlk gene in the individuals with MDS. Dlk could be the first candidate molecule to differentiate MDS from AML. The proposal is made that microarray analysis with the Blast Bank samples is an efficient approach to extract transcriptome data of clinical relevance for a wide range of hematologic disorders.

Identification of human GATA-2 gene distal IS exon and its expression in hematopoietic stem cell fractions.

Transcription factor GATA-2 is essential for the proper function of hematopoietic stem cells and progenitors. Two first exons/promoters have been found in the mouse GATA-2 gene, and a distal IS promoter shows activity specific to hematopoietic progenitors and neural tissues. To ascertain whether the two-promoter system is also utilized in the human GATA-2 gene, we isolated and analyzed a P1 phage clone containing this gene. The nucleotide sequence of the human GATA-2 gene 5' flanking region was determined over 10 kbp, and a human IS exon was identified in the locus through sequence comparison analysis with that of the mouse GATA-2 IS exon. RNA blotting and reverse-transcribed PCR analyses identified a transcript that starts from the IS exon in human leukemia-derived cell lines. The IS-originated transcript was also identified in CD34-positive bone marrow and cord blood mononuclear cells, which are recognized as clinically important hematopoietic stem cell-enriched fractions. Phylogenic comparison of the human and mouse GATA-2 gene sequences revealed several regions in the locus that exhibit high sequence similarity. These results demonstrate that the GATA-2 gene regulatory machinery is conserved among vertebrates. The fact that the human IS promoter is active in the hematopoietic stem cell/progenitor fraction may be an important clue for the design of a vector system that can specifically express various genes in hematopoietic stem cells and progenitors.

Identification of a Novel Polyproline Recognition Site in the Cytoskeletal Associated Protein, Proline Serine Threonine Phosphatase Interacting Protein

Protein-protein interactions are often mediated by the recognition of proline-rich domains by SH3 or WW modules. Previously, we demonstrated that the PEST-type protein-tyrosine phosphatase, PTP HSCF (hematopoietic stem cell fraction), bound to a novel cytoskeletal associated protein, proline serine threonine phosphatase interacting protein (PST PIP), via an interaction between the proline-rich COOH terminus of the PTP and a site within the putative coiled-coil domain of PST PIP. Here we describe a more detailed analysis of this interaction. Earlier data suggested that the NH2 terminus of PST PIP was important for binding to the phosphatase, and deletion of the NH2-terminal 50 amino acids of the PST PIP resulted in an apparently misfolded protein that was incapable of binding PTP HSCF. To examine the region involved with binding to PTP HSCF, alanine-scanning mutants were produced at intervals throughout PST PIP. This analysis demonstrated that a tryptophan at position 232 was essential for binding in vitro. Transfection experiments demonstrated that the Trp232 mutant protein was capable of association with the cortical cytoskeleton but was not bound to PTP HSCF in vivo. Alanine scanning of a peptide derived from the COOH-terminal proline-rich domain of PTP HSCF revealed that a subset of prolines, as well as other residues, was required for efficient binding to PST PIP, and introduction of alanines at some of these positions in the protein resulted in decreased binding to PST PIP in vitro and in vivo. Analysis of in vivo tyrosine phosphorylation of the Trp232 mutant of PST PIP in the presence of v-Src revealed that this protein was phosphorylated more efficiently than the wild-type molecule. Thus, the interaction between PTP HSCF and PST PIP is mediated by a novel site in the cytoskeletal associated protein which interacts with residues within the proline-rich COOH terminus of the phosphatase.

PSTPIP: a tyrosine phosphorylated cleavage furrow-associated protein that is a substrate for a PEST tyrosine phosphatase.

We have investigated proteins which interact with the PEST-type protein tyrosine phosphatase, PTP hematopoietic stem cell fraction (HSCF), using the yeast two-hybrid system. This resulted in the identification of proline, serine, threonine phosphatase interacting protein (PSTPIP), a novel member of the actin- associated protein family that is homologous to Schizosaccharomyces pombe CDC15p, a phosphorylated protein involved with the assembly of the actin ring in the cytokinetic cleavage furrow. The binding of PTP HSCF to PSTPIP was induced by a novel interaction between the putative coiled-coil region of PSTPIP and the COOH-terminal, proline-rich region of the phosphatase. PSTPIP is tyrosine phosphorylated both endogenously and in v-Src transfected COS cells, and cotransfection of dominant-negative PTP HSCF results in hyperphosphorylation of PSTPIP. This dominant-negative effect is dependent upon the inclusion of the COOH-terminal, proline-rich PSTPIP-binding region of the phosphatase. Confocal microscopy analysis of endogenous PSTPIP revealed colocalization with the cortical actin cytoskeleton, lamellipodia, and actin-rich cytokinetic cleavage furrow. Overexpression of PSTPIP in 3T3 cells resulted in the formation of extended filopodia, consistent with a role for this protein in actin reorganization. Finally, overexpression of mammalian PSTPIP in exponentially growing S. pombe results in a dominant-negative inhibition of cytokinesis. PSTPIP is therefore a novel actin-associated protein, potentially involved with cytokinesis, whose tyrosine phosphorylation is regulated by PTP HSCF.

Phenotypic and Functional Changes Induced at the Clonal Level in Hematopoietic Stem Cells After 5-Fluorouracil Treatment

AbstractA significant fraction of hematopoietic stem cells (HSCs) have been shown to be resistant to the effects of cytotoxic agents such as 5-fluorouracil (5-FU), which is thought to eliminate many of the rapidly dividing, more committed progenitors in the bone marrow and to provide a relatively enriched population of the most primitive hematopoietic progenitor cells. Although differences between 5-FU–enriched progenitor populations and those from normal bone marrow have been described, it remained unclear if these differences reflected characteristics of the most primitive stem cells that were revealed by 5-FU, or if there were changes in the stem-cell population itself. Here, we have examined some of the properties of the stem cells in the bone marrow before and after 5-FU treatment and have defined several activation-related changes in the stem-cell population. We found that long-term reconstituting stem cells decrease their expression of the growth factor receptor c-kit by 10-fold and increase their expression of the integrin Mac-1 (CD11b). These changes begin as early as 24 hours after 5-FU treatment and are most pronounced within 2 to 3 days. This activated phenotype of HSCs isolated from 5-FU–treated mice is similar to the phenotype of stem cells found in the fetal liver and to the phenotype of transiently repopulating progenitors in normal bone marrow. We found that cell cycle is induced concomitantly with these physical changes, and within 2 days as many as 29% of the stem-cell population is in the S/G2/M phases of the cell cycle. Furthermore, when examined at a clonal level, we found that 5-FU did not appear to eliminate many of the transient, multipotent progenitors from the bone marrow that were found to be copurified with long-term repopulating, activated stem cells. These results demonstrate the sensitivity of the hematopoietic system to changes in its homeostasis and correlate the expression of several important surface molecules with the activation state of HSCs.

Phenotypic and Functional Changes Induced at the Clonal Level in Hematopoietic Stem Cells After 5-Fluorouracil Treatment

A significant fraction of hematopoietic stem cells (HSCs) have been shown to be resistant to the effects of cytotoxic agents such as 5-fluorouracil (5-FU), which is thought to eliminate many of the rapidly dividing, more committed progenitors in the bone marrow and to provide a relatively enriched population of the most primitive hematopoietic progenitor cells. Although differences between 5-FU–enriched progenitor populations and those from normal bone marrow have been described, it remained unclear if these differences reflected characteristics of the most primitive stem cells that were revealed by 5-FU, or if there were changes in the stem-cell population itself. Here, we have examined some of the properties of the stem cells in the bone marrow before and after 5-FU treatment and have defined several activation-related changes in the stem-cell population. We found that long-term reconstituting stem cells decrease their expression of the growth factor receptor c-kit by 10-fold and increase their expression of the integrin Mac-1 (CD11b). These changes begin as early as 24 hours after 5-FU treatment and are most pronounced within 2 to 3 days. This activated phenotype of HSCs isolated from 5-FU–treated mice is similar to the phenotype of stem cells found in the fetal liver and to the phenotype of transiently repopulating progenitors in normal bone marrow. We found that cell cycle is induced concomitantly with these physical changes, and within 2 days as many as 29% of the stem-cell population is in the S/G2/M phases of the cell cycle. Furthermore, when examined at a clonal level, we found that 5-FU did not appear to eliminate many of the transient, multipotent progenitors from the bone marrow that were found to be copurified with long-term repopulating, activated stem cells. These results demonstrate the sensitivity of the hematopoietic system to changes in its homeostasis and correlate the expression of several important surface molecules with the activation state of HSCs.

Thrombopoietin: more than a lineage-specific megakaryocyte growth factor.

In the past two years thrombopoietin (TPO) has been cloned, its effects on cells of the megakaryocytic lineage have been described in detail and its use in clinical settings of myelosuppressive therapy has begun. Moreover, the mechanisms by which the hormone binds to its receptor have been studied in detail, and the intracellular pathways employed during TPO signaling have been extensively explored. Although most workers in the field predicted that TPO would be lineage-specific, with physiologic effects limited to megakaryocytes and platelets, several features of Mpl biology suggest its influence on hematopoiesis may be more widespread than initially anticipated. To test this possibility, we and others have begun to explore whether TPO affects development of the hematopoietic stem cell. In suspension culture, TPO alone can support the survival of a fraction of hematopoietic stem cells but does not lead to their proliferation. However, in combination with interleukin 3 or stem cell factor, TPO accelerates hematopoietic stem cell entry into the cell cycle over that seen with these early-acting cytokines alone, increases the number of subsequent cell divisions per unit time and results in the output of far greater numbers of colony-forming cells of all lineages. Conclusions from these in vitro studies are supported by two types of in vivo experiments. The administration of TPO to either normal or myelosuppressed animals causes an expansion of colony-forming unit (CFU)-megakaryocyte, CFU-granulocyte-macrophage, CFU-granulocyte/erythroid/macrophage/megakaryocyte and BFU-E, and elimination of TPO or its receptor by genetic engineering results in a substantial decrease in the numbers of these progenitors in both the marrow and spleen. It is thus becoming clear that the effects of TPO extend beyond that of a megakaryocyte-specific factor and suggest that the hematopoietic effects of administration of the hormone may be greater than initially anticipated.

Mathematical models of marrow cell kinetics: Differential effects of protracted irradiations on stromal and stem cells in mice

It is known that hematopoiesis is supported by bone-marrow stem cells, but those cells must seed and grow on a stromal microenvironment. Typically, studies have shown that a surviving fraction of about 30 hematopoietic stem cells (HSCs) (i.e., about 0.04%) correspond to the LD 50, although other studies have shown that marrow can repopulate from a single viable cell under strong regiments of antibiotics and infusions of irradiated blood elements. Purpose : This paper describes comparisons between our results (from maximum-likelihood estimation techniques for cellular damage, repair, and compensatory repopulation) and published experimental data on marrow stromal cells. Methods and Materials : After biophysical consideration of the rate constants that were derived by maximizing the likelihood function (a consideration necessary to extend the model to cell populations not indicated by the model as “critical” for recovery), the rate constants for cellular damage to stem cells are fitted to experimental data. Rate constants for repair and proliferation of stem cells are assigned based on published data on repair/proliferation halftimes, and these assignments affect the evaluation of the rate constants for cellular damage. From the two models, that is one for “critical” cells (having radiosensitive and repopulation characteristics similar to stromal cells) and another for stem cells, effects on two cell populations of different radiosensitivities and repopulation rates can be demonstrated for complex schedules of protracted irradiations which could reduce either cell population below a critical need for marrow repopulation. Results : Our analysis of animal mortality data has indicated that recovery of an animal from potentially lethal irradiation is dominantly regulated by cells with survival and repopulation characteristics similar to those of stroma cells. Conclusion : In contrast to the surviving fraction of hematopoietic stem cells, it appears that the probability of an animal's recovery is high if the “critical” population of cells is above 1% (our “best” maximum likelihood estimate, from mouse data, with the corresponding lower confidence bound at about 0.2%). Of course, a few stem cells—perhaps only one—must maintain a potential for repopulation of blood and marrow.

Sequential Analysis of Hematopoietic Reconstitution Achieved By Transplantation of Hematopoietic Stem Cells

Abstract We confirmed that murine hematopoietic stem cells express the c-kit molecule but not lymphohematopoietic lineage markers. These lineage marker-negative c-kit-positive (Lin- c-kit+) cells were further divided according to the uptake of rhodamine-123 (Rh-123). Approximately 1,000 Lin- c-kit+ rhodamine-123dull cells, which contained 4.0 +/- 1.3 and 12.5 +/- 1.9 day 8 and day 12 spleen colony-forming units (CFU-S), respectively, rescued the 100% of lethally irradiated mice. One third of these cells formed colonies in the presence of interleukin-3 plus erythropoietin. The time course of the hematopoietic reconstitution of this primitive hematopoietic stem cell fraction was investigated by using Ly-5 congenic mice. Although myeloid cells and B lymphocytes were detected in the peripheral blood 2 to 3 weeks after transplantation, T lymphocytes were not detected until 4 weeks after transplantation. It is generally assumed that myeloid cells and B lymphocytes grow in the bone marrow and that T lymphocytes must pass through the thymus. For the first 2 to 3 weeks after transplantation, donor-type T lymphocytes were not dominant in the thymus, and most donor type cells were CD4/CD8 double-negative or double-positive (including CD4low and CD8low). Four weeks after transplantation, donor-type T lymphocytes were dominant and the ratio of CD4/CD8 cells had recovered to the normal pattern. However, significant numbers of T lymphocytes were detected in the peripheral blood at this stage. Sequential analysis of hematopoietic reconstitution from primitive stem cells demonstrates that myeloid and B-lymphoid lineages occurred earlier than that of the T-lymphoid lineages.

Sequential analysis of hematopoietic reconstitution achieved by transplantation of hematopoietic stem cells

We confirmed that murine hematopoietic stem cells express the c-kit molecule but not lymphohematopoietic lineage markers. These lineage marker-negative c-kit-positive (Lin- c-kit+) cells were further divided according to the uptake of rhodamine-123 (Rh-123). Approximately 1,000 Lin- c-kit+ rhodamine-123dull cells, which contained 4.0 +/- 1.3 and 12.5 +/- 1.9 day 8 and day 12 spleen colony-forming units (CFU-S), respectively, rescued the 100% of lethally irradiated mice. One third of these cells formed colonies in the presence of interleukin-3 plus erythropoietin. The time course of the hematopoietic reconstitution of this primitive hematopoietic stem cell fraction was investigated by using Ly-5 congenic mice. Although myeloid cells and B lymphocytes were detected in the peripheral blood 2 to 3 weeks after transplantation, T lymphocytes were not detected until 4 weeks after transplantation. It is generally assumed that myeloid cells and B lymphocytes grow in the bone marrow and that T lymphocytes must pass through the thymus. For the first 2 to 3 weeks after transplantation, donor-type T lymphocytes were not dominant in the thymus, and most donor type cells were CD4/CD8 double-negative or double-positive (including CD4low and CD8low). Four weeks after transplantation, donor-type T lymphocytes were dominant and the ratio of CD4/CD8 cells had recovered to the normal pattern. However, significant numbers of T lymphocytes were detected in the peripheral blood at this stage. Sequential analysis of hematopoietic reconstitution from primitive stem cells demonstrates that myeloid and B-lymphoid lineages occurred earlier than that of the T-lymphoid lineages.

Effect of polynosinic acid-polycytidylic acid on colony-forming capacity of the hematopoietic stem cell fraction during allogeneic inhibition

Partial restoration of colony-forming ability was found in a graft of parental (C57BL/6) bone marrow in a (CBAxC57BL/6)F1 hydrid recipient irradiated in a dose of 800 rad, after injection of a polyI-poly C preparation. Colony formation was increased to the same extent as after addition of thymus cells of the same parent. In both cases the number of colonies per spleen was more than doubled compared with the control. In the syngeneic system thymus cells and the polyI-polyC preparation had no effect on the number of splenic colonies formed. If the number of transplanted bone marrow cells remained constant (5·105), an increase in the dose of polyI-polyC (from 50 to 100 μg) and of thymus cells (from 4·106 to 8·106) did not lead to any increase in the efficiency of colony formation.