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PubMed1983

Can hypertransfusion attenuate myelosuppression associated with combination chemotherapy in patients with inoperable bronchogenic carcinoma? A report of a randomised, controlled study.

Based on the concept of "stem-cell competition," hypertransfusion has been shown to attenuate the leucopenia associated with chemotherapy in children with leukemia. We conducted a randomized, controlled study of hypertransfusion in 25 patients with inoperable lung cancer who received combination chemotherapy consisting of methotrexate, adriamycin, cyclophosphamide, and CCNU (Lomustine) (MACC scheme). Twelve patients in the hypertransfusion group were given red cell transfusion to a hemoglobin value greater than or equal to 17 gm/dl prior to each chemotherapy cycle. The two groups of patients were comparable in age, cell type, extent of disease, performance status, and initial hemoglobin level and blood counts. The mean fall in granulocyte count was greater for control group (3.76 X 10(9)/liter) than for hypertransfusion group (3.27 X 10(9)/liter), and the mean fall in platelet count was greater for control group (53.84 X 10(9)/liter) than for hypertransfusion group (35.83 X 10(9)/liter), although the differences did not reach statistical significance (p greater than 0.05) partly because our MACC scheme was probably not sufficiently myelosuppressive to bring about a difference in the two groups. Granulocytopenia-associated infections were infrequent in both groups: two episodes in 37 cycles of chemotherapy in the hypertransfusion group and three episodes in 43 cycles in the control group. Hypertransfusion was simple and safe, and the encouraging trend towards less marked myelosuppression in our hypertransfused group would warrant further studies using more intensive and myelosuppressive combination chemotherapy regimens.

Adult↗
PubMed1978

Platelet production in hypoxic and RBC-transfused mice.

Platelet production rates were studied in hypoxic, red blood cell (RBC) transfused, and normal mice. In addition, platelet depletion was induced in some of the mice by injection of rabbit anti-mouse platelet serum (RAMPS) to stimulate platelet production. Hypoxia alone caused an increase in haematocrit and platelet count at 1-3 d, followed by a decrease in platelet counts to below normal values at 6-7 d. On the otherhand, RBC transfusion caused increase haematocrit and decreased platelet count of mice at 1-4 d, with a return of platelet counts to normal by 5-6 d. Normal mice and mice transfused with RBC responded to platelet depletion with rebound-thrombocytosis with maximum platelet production 3-5 d later and elevated platelet counts on days 5-6. However, platelet production in platelet-depleted mice exposed to hypoxia was less marked, and platelet counts did not reach normal levels. The data are consistent with the hypothesis that hypoxia causes thrombocytopenia by stem cell competition between erythroid and megakaryocytic cell lines and/or inhibition of thrombopoietin production.

Animals↗
PubMed1999

The effect of recombinant human erythropoietin on platelet counts is strongly modulated by the adequacy of iron supply.

The effect of recombinant human erythropoietin (rHuEpo) on megakaryopoiesis remains controversial. Treatment with rHuEpo in renal failure patients has been associated with a slight elevation of platelet counts. In animal studies, high doses of rHuEpo produced an increase of platelet counts followed by a gradual return to normal after 7 to 15 days or even a substantial degree of thrombocytopenia. However, because iron deficiency is also known to be associated with thrombocytosis, (functional) iron deficiency during rHuEpo could be contributing to these observations. We investigated the impact of iron supply on changes in platelet counts induced by rHuEpo. Rats were either fed normal food (normal rats) or received 1% carbonyl iron for 2 weeks or 3 months, as well as during the experiment, to achieve iron supplementation or overload, respectively. Rats of all three categories then received daily intravenous injections of rHuEpo (10, 50, or 150 U) or normal saline (0 U) for 20 days. With 0 to 10 U rHuEpo, platelets remained stable. In normal rats receiving 50 to 150 U rHuEpo, platelets increased to 120% to 140% of baseline at 4 to 12 days to level off at 120% at 16 to 20 days. This response was less sustained in splenectomized animals. Iron-supplemented rats receiving 50 to 150 U rHuEpo also increased platelets initially, but the peak was at day 4, followed by a gradual return to baseline and even a moderate thrombocytopenia later on. Iron-overloaded rats receiving 50 to 150 U rHuEpo also had increased platelets at day 4, but the duration of platelet increase was shorter, and they experienced a more pronounced degree of thrombocytopenia in proportion to the dose of rHuEpo. Because the early elevation of platelets was of larger magnitude than hematocrit changes, it is unlikely that it could be accounted for by shrinkage of plasma volume. Because it was observed in all three iron conditions, there appears to be some direct positive effect of rHuEpo on platelet production. However, after this transient effect, expanded erythropoiesis appears to exert a negative impact upon platelet production. Secondary thrombocytopenia was not related to splenic pooling, and its very slow correction after cessation of rHuEpo therapy is not compatible with changes in platelet survival. Rather, it is consistent with stem cell competition between erythroid and megakaryocytic development. However, this secondary thrombocytopenia is masked by (functional) iron deficiency in rats not receiving an adequate iron supply from food or stores.

Animals↗
PubMed1999

Erythropoietin and platelet production.

BACKGROUND AND OBJECTIVE: Erythropoietin (Epo) is the primary growth factor for the red cell lineage but treatment with recombinant human Epo (rHuEpo) has been shown to increase platelet counts. In several animal species treatment with rHuEpo stimulated platelet production, but platelet counts tended to normalize after 1-2 weeks and large, chronic doses even caused thrombocytopenia. This paper aims to review the evidence about the effects of Epo on megakaryopoiesis. INFORMATION SOURCES: I examined the literature published in journals covered by Medline(R)â concerning the effects of Epo, hypoxia and iron deficiency on megakaryopoiesis and platelets. The reference list of each article was reviewed to try to identify further contributions. STATE OF THE ART: In vivo data have shown that moderate Epo stimulation, i.e. that produced by standard doses of rHuEpo, short-term hypoxia or moderate iron deficiency, causes a moderate elevation of platelet counts, whereas intense Epo stimulation, as produced by high doses of rHuEpo, prolonged hypoxia or severe iron deficiency, causes some degree of thrombocytopenia. In the latter case, there appears to be a diphasic response to Epo, the initial positive response (a stimulation of platelet production) being followed by thrombocytopenia. Contrarily to the thrombocytopenia due to increased platelet destruction induced by other growth factors, Epo-induced thrombocytopenia is the result of an inhibition of platelet production. CONCLUSION AND PERSPECTIVE: Stem-cell competition between erythroid and platelet precursors appears to be the cause of these phenomena in situations of prolonged, intense stimulation by Epo. In vitro data support the existence of a common erythrocytic and megakaryocytic precursor. It remains to be determined whether these effects of rHuEpo are a result of the dose itself or of the magnitude of the erythropoietic effect of that dose. It is not known whether a lower dose given in a patient with decreased marrow function would bring about the same biological effects as those induced by high doses of rHuEpo in the presence of a normal marrow function. Caution should be exercised before using high doses of hematopoietic growth factors.

Blood Platelets↗
PubMed1992

Comparison of platelet production in two strains of mice with different modal megakaryocyte DNA ploidies after exposure to hypoxia.

Thrombocytopenia develops with prolonged exposure to hypoxia. Although decreases in megakaryocyte numbers due to hypoxia have been well documented, the effects of hypoxia on megakaryocyte DNA content have not been reported. In this study, megakaryocytopoiesis and platelet production were compared in both C3H mice (whose megakaryocyte modal ploidy class is 32N) and C57/BL mice (whose modal ploidy class is 16N), by enclosure in cages covered with silicone-rubber membranes. After equilibration, O2 levels inside the cages were 6%-7%. Hematocrits, platelet counts, platelet sizes, percent 35S incorporation into platelets, megakaryocyte size and number, and megakaryocyte DNA content of mice were measured before and at various days after hypoxia. Although hematocritis increased and platelet counts decreased in both strains of mice with time in hypoxic chambers, megakaryocyte and platelet responses of C3H mice differed from those of C57/BL mice in several respects; hematocrits of C3H mice were higher and platelet counts were lower than those in C57/BL mice. C3H mice produced larger platelets than C57/BL mice in response to hypoxia. Total circulating platelet counts (TCPC) and total circulating platelet masses (TCPM) of both mouse strains showed similar biphasic responses, that is, elevated TCPC and TCPM on days 2-4 and decreased values after 6-14 days of hypoxia. However, hypoxic C3H mice had lower TCPC on days 4-14 and lower TCPM on days 10-14 of hypoxia than C57/BL mice. Both C3H and C57/BL mice had decreased megakaryocyte numbers at 6-10 days of hypoxia, but only C3H mice had decreased numbers of megakaryocytes at day 14. Elevated megakaryocyte size was observed in both mouse strains at day 14 of hypoxia. However, after hypoxia, C3H mice showed a greater depression in megakaryocyte number and a larger increase in megakaryocyte sizes than did C57/BL mice. C3H mice maintained 32N as the modal megakaryocyte DNA content through day 10 of hypoxia, but 64N was the modal megakaryocyte DNA content at day 14; 16N remained the modal megakaryocyte DNA content in hypoxic C57/BL mice. Hypoxic C3H mice had an increase in 16N megakaryocytes after 6 days of hypoxia, followed by an increase in the proportion of 64N cells at 14 days compared to values of untreated C3H control mice. Hypoxic C57/BL mice had an increased proportion of 16N cells at 6 days but a decreased proportion of 32N cells at 14 days. These studies demonstrate that the decreased platelet production resulting from prolonged exposure to hypoxia is primarily the result of decreased differentiation of hematopoietic precursors into the megakaryocyte lineage rather than decreased megakaryocyte DNA content, because higher ploidy classes actually increase as thrombocytopenia becomes more severe. Stem cell competition could explain the findings of reduced platelet production and increased red blood cell production in both strains of mice after exposure to hypoxia.

Animals↗
PubMed1989

Enhancement by dimethyl myleran of donor type chimerism in murine recipients of bone marrow allografts.

A major problem in using murine models for studies of bone marrow allograft rejection in leukemia patients is the narrow margin in which graft rejection can be analyzed. In mice irradiated with greater than 9 Gy total body irradiation (TBI) rejection is minimal, whereas after administration of 8 Gy TBI, which spares a significant number of clonable T cells, a substantial frequency of host stem cells can also be detected. In current murine models, unlike in humans, bone marrow allograft rejection is generally associated with full autologous hematopoietic reconstitution. In the present study, we investigated the effect of the myeloablative drug dimethyl myleran (DMM) on chimerism status following transplantation of T cell-depleted allogenic bone marrow (using C57BL/6 donors and C3H/HeJ recipients, conditioned with 8 Gy TBI). Donor type chimerism 1 to 2 months post-transplant of 1 to 3 x 10(6) bone marrow cells was markedly enhanced by using DMM one day after TBI and prior to transplantation. Conditioning with cyclophosphamide instead of DMM, in combination with 8 Gy TBI, did not enhance engraftment of donor type cells. Artificial reconstitution of T cells, after conditioning with TBI plus DMM, by adding mature thymocytes, or presensitization with irradiated donor type spleen cells 1 week before TBI and DMM, led to strong graft rejection and consequently to severe anemia. The anti-donor responses in these models were proportional to the number of added T cells and to the number of cells used for presensitization, and they could be neutralized by increasing the bone marrow inoculum. These results demonstrate the potential of DMM to facilitate engraftment in unsensitized mice in which the host stem cells may compete with donor type cells; the use of DMM to create models in which mechanisms of immune rejection can be studied without interference due to stem cell competition; and that bone marrow allograft rejection may be overcome by increasing the bone marrow inoculum in these stringent models.

Animals↗
PubMed1983

Cyclic neutropenia: amplification of granulocyte oscillations by lithium and long-term suppression of cycling by plasmapheresis.

A patient with well documented cyclic neutropenia (CN) was given chronic lithium therapy as well as a short course of plasmapheresis with therapeutic intention. While on lithium therapy, an increase in the amplitude of the granulocyte oscillations was observed, but recurrent agranulocytotic periods persisted. A 2-wk course of plasmapheresis (total exchange 17 liters) resulted in a gradual decrease of granulocyte oscillations, with the granulocyte count remaining above 500/cu mm at all times, and the patient became asymptomatic. The improvements in the clinical state of the patient have now persisted for more than 9 mo. Quantitative analysis of the oscillation period and the phase lags between peak counts of the hematologic cell lines revealed a 20-day cycle for granulocytes, monocytes, reticulocytes, and platelets, but not for lymphocytes. The observed phase lags, together with the literature values for the intramarrow maturation times of the hematologic cell lines, suggest that the feedback regulation abnormality, believed to be at the basis of CN, primarily affects the stem cell that is still capable of differentiating into granulocytes and monocytes, but that the oscillations of reticulocytes and platelets are the result of a stem cell competition for uncommitted precursor cells by the primarily oscillating granulocyte and monocyte production.

Aged↗
PubMed1977

Effects of chronic hypoxia on platelet production in mice.

Platelet production, platelet count, and RBC count were measured in mice after enclosure in silicone-rubber membrane cages. Results indicate that enclosing C3h mice in hypoxia chambers for 2 weeks caused increased RBC count and decreased platelet production leading to thrombocytopenia. Splenectomized C3H mice placed in hypoxia chambers also showed decreased platelet count and platelet production when compared to splenectomized ambient air control mice. Injection of rabbit anti-mouse-platelet serum (RAMPS) caused an immediate thrombocytopenia with rebound-thrombocytosis 5 days later in normal mice and mice transfused with RBC. However, mice exposed to hypoxia and given RAMPS injections exhibited marked inhibition of platelet production. Although indirect, these findings suggest that hypoxia-induced erythrocythemia causes thrombocytopenia by stem cell competition between the erythroid and megakaryocytic cell lines.

Animals↗
PubMed2001

HOXB4 overexpression mediates very rapid stem cell regeneration and competitive hematopoietic repopulation.

OBJECTIVE: Hox transcription factors have emerged as important regulators of hematopoiesis. In particular, we have shown that overexpression of HOXB4 in mouse bone marrow can greatly enhance the level of hematopoietic stem cell (HSC) regeneration achieved at late times (> 4 months) posttransplantation. The objective of this study was to resolve if HOXB4 increases the rate and/or duration of HSC regeneration, and also to see if this enhancement was associated with impaired production of end cells or would lead to competitive reconstitution of all compartments. METHODS: Retroviral vectors were generated with the GFP reporter gene +/- HOXB4 to enable the isolation and direct tracking of transduced cells in culture or following transplantation. Stem cell recovery was measured by limit dilution assay for long-term competitive repopulating cells (CRU). RESULTS: HOXB4-overexpressing cells have enhanced growth in vitro, as demonstrated by their rapid dominance in mixed cultures and their shortened population doubling time. Furthermore, HOXB4-transduced cells have a marked competitive repopulating advantage in vivo in both primitive and mature compartments. CRU recovery in HOXB4 recipients was extremely rapid, reaching 25% of normal by 14 days posttransplant or some 80-fold greater than control transplant recipients, and attaining normal numbers by 12 weeks. Mice transplanted with even higher numbers of HOXB4-transduced CRU regenerated up to but not beyond the normal CRU levels. CONCLUSIONS: HOXB4 is a potent enhancer of primitive hematopoietic cell growth, likely by increasing self-renewal probability but without impairing homeostatic control of HSC population size or the rate of production and maintenance of mature end cells.

Animals↗
PubMed1995

Long-term repopulating abilities of enriched fetal liver stem cells measured by competitive repopulation.

To characterize hematopoietic cell biology, many investigators have used protocols that enrich for primitive hematopoietic stem cells (PHSC). In this study, we quantified the long-term repopulating ability (LTRA) of enriched and discarded fractions of PHSC from day-14 murine fetal liver using the competitive repopulation assay. We fractionated populations of fetal cells using the antigenic markers AA4.1+, AA4.1+/Sca+, and AA4.1+/Linlow/Sca+. Differentiating and repopulating abilities of each of these populations were directly compared using competitive repopulation. Adult bone marrow was mixed with fetal cell fractions from congenic donors having genetically distinguishable markers, and mixtures were given to irradiated recipients. Differentiating and repopulating abilities of the enriched donor cells were measured by the proportions of myeloid and lymphoid cells having donor markers that repopulated the recipients. LTRA was found primarily in the AA4.1+ and AA4.1+/Sca+ subpopulations. Further fractionation of the AA4.1+ cells to derive an AA4.1+/Linlow/Sca+ fraction showed that virtually all of the long-term stem cell activity was found in this subpopulation. These cells were 1400- to 1600-fold enriched in long-term functional ability compared to fresh marrow. This very high multilineage repopulating ability per cell was directly measured using a long-term functional assay in vivo. Importantly, the measured repopulating ability for AA4.1+/Linlow/Sca+ cells was about five-fold less than expected from the fraction of cells enriched and remained two- to three-fold less even after compensating for repopulating ability in discarded fractions. This illustrates that long-term functional abilities of enriched PHSC cannot be estimated from fractions enriched but should be quantitatively assayed.

Aging↗
PubMed1989

Isolation in a single step of a highly enriched murine hematopoietic stem cell population with competitive long-term repopulating ability.

A simple procedure is described for the quantitation and enrichment of murine hematopoietic cells with the capacity for long-term repopulation of lymphoid and myeloid tissues in lethally irradiated mice. To ensure detection of the most primitive marrow cells with this potential, we used a competitive assay in which female recipients were injected with male "test" cells and 1 to 2 x 10(5) "compromised" female marrow cells with normal short-term repopulating ability, but whose long-term repopulating ability had been reduced by serial transplantation. Primitive hematopoietic cells were purified by flow cytometry and sorting based on their forward and orthogonal light-scattering properties, and Thy-1 and H-2K antigen expression. Enrichment profiles for normal marrow, and marrow of mice injected with 5-fluorouracil (5-FU) four days previously, were established for each of these parameters using an in vitro assay for high proliferative potential, pluripotent colony-forming cells. When all four parameters were gated simultaneously, these clonogenic cells were enriched 100-fold. Both day 9 and day 12 CFU-S were copurified; however, the purity (23%) and enrichment (75-fold) of day 12 CFU-S in the sorted population was greater with 5-FU-treated cells. Five hundred of the sorted 5-FU marrow cells consistently repopulated recipient lymphoid and myeloid tissues (greater than 50% male, 1 to 3 months post-transplant) when co-injected with 1 to 2 x 10(5) compromised female marrow cells, and approximately 100 were sufficient to achieve the same result in 50% of recipients under the same conditions. This relatively simple purification and assay strategy should facilitate further analysis of the heterogeneity and regulation of stem cells that maintain hematopoiesis in vivo.

Animals↗
PubMed1982

The competitive ability of stem cells from mice with Hertwig's anemia.

Mice homozygous for the gene, an, have a macrocytic, normochromic anemia. In this report, attempts have been made to cure Hertwig's anemia (an/an) by injecting genetically normal (+/+) stem cells. The anemia of unirradiated an/an mice was alleviated but not completely cured by injection of as many as 3 X 10(7) +/+ bone marrow cells. Lethal irradiation of the an/an recipients was necessary before injections of 10(7) +/+ marrow cells were effective in normalizing the blood parameters. The inability to achieve normal blood values without first destroying the host's own stem cells suggested that the indigenous an/an cells compete effectively with injected +/+ cells. This hypothesis was tested by injecting varying numbers of stem cells from C57BL/6J-+/+ mice, together with stem cells from either WBB6F1-an/an or, as controls, from their WBB6F1-+/+ littermates, into lethally irradiated hosts. The C57BL/6J and WBB6F1 mice have electrophoretically distinguishable hemoglobins. The an/an cells are able to compete in the repopulation of the host hematopoietic tissue as shown by the presence of WBB6F1 hemoglobin in the recipients. The cells from mice with Hertwig's anemia, however, do not compete as effectively as do the same number of cells from the +/+ littermates. These results indicate that the pluripotent hematopoietic stem cells of an/an mice are reduced in number, seeding capacity, or proliferative potential.

Anemia, Macrocytic↗
PubMed1993

Competitive repopulation in leukemic and normal bone marrow.

The success of chemotherapy in leukemias in which the marrow appears entirely replaced by leukemic cells must be due to the persistence of some normal stem cells. The implied competition between leukemic and normal stem cells is thus akin to the competition between donor and host cells in irradiated animals. A review of that competition points to the importance of the quantitative relationships between the competing stem cells, even when one of the competing stem cell clones has a proliferative advantage. Pursuing that analogy, it is suggested that stimulating the surviving normal stem cells by appropriate combinations of cytokines may be of therapeutic benefit, once the tumor load has been reduced by chemotherapy. Complete eradication of leukemic cells may not be necessary, if surviving normal cells could gain ascendancy over the residual leukemic cell clones.

Animals↗
PubMed2002

Donor chimerism and stem cell function in a murine congenic transplantation model after low-dose radiation conditioning: effects of a retroviral-mediated gene transfer protocol and implications for gene therapy.

OBJECTIVE: We investigated low-dose radiation conditioning for the transplantation of retrovirus-transduced cells in a C57Bl6/J murine model. MATERIALS AND METHODS: The effect of low-dose radiation on stem cell function was investigated using a competitive repopulation assay. Stem cell function of marrow cells that underwent a retroviral-mediated gene transfer (RMGT) protocol was examined by this assay, and donor chimerism of these cells when transplanted into 160-cGy conditioned syngeneic hosts was compared to fresh marrow. RESULTS: Irradiation with 300 or 160 cGy substantially decreased stem cell function as measured by competitive repopulation. Animals conditioned with 160 cGy and transplanted with 20 x 10(6) fresh marrow cells permitted donor cell engraftment of 53.6% +/- 11.4% 6 months after transplant compared to 100% donor cell engraftment after 1100 cGy irradiation. Lymphoid and myeloid engraftment did not significantly differ from total engraftment in submyeloablated hosts. When transplanted into lethally irradiated hosts, the competitive repopulating activity of marrow treated with a single dose of 5-fluorouracil followed by ex vivo culture according to a standard RMGT protocol was equal to 5-fluorouracil-only treated marrow. However, cells treated with 5-fluorouracil or 5-fluorouracil plus ex vivo culture for RMGT repopulated less well than fresh marrow cells in 160 cGy conditioned hosts. CONCLUSIONS: Low-dose irradiation decreases host stem cell function, allowing engraftment of both fresh and RMGT protocol-treated marrow, although the engraftment of 5-fluorouracil-treated cells was reduced at least two-fold, and 5-fluorouracil plus RMGT protocol-treated cells at least three-fold, compared to fresh marrow. Modification of current RMGT protocols may be important for optimizing engraftment under these conditions.

Animals↗
PubMed1977

[Inactivation of stem cells by allogenic lymphocytes: competition between T1- and T2-subpopulations].

Transplantation of the bone marrow cells with allogeneic T-lymphocytes to the irradiated hosts was accompanied by inactivation of the stem elements of the graft. The lymph node cells of T-mice (those deprived of B cells) were more active than the spleen cells of these mice. The stem cells inactivation was weakly expressed or absent in case of a combined acti-n of T-cells from the lymph nodes and the spleen.

Animals↗
PubMedDate not supplied

The Cooperative Care model: an innovative approach to deliver blood and marrow stem cell transplant care.

Competition among healthcare institutions, the need to improve outcomes, and the desire to decrease costs have motivated blood and marrow stem cell transplant centers to develop innovative care models. In an effort to meet these challenges, a major midwestern medical center adapted the transplant process to the outpatient setting. This transition created greater educational and care demands for patients and families. To address these demands and provide improved accommodations and amenities for patients and families, the center adopted an innovative model of care, Cooperative Care, for transplant recipients. Cooperative Care embraces patients and families as key members of the healthcare team. A family member serves as a primary caregiver for the patient during the acute inpatient phase of the transplant. Care becomes more personal and individualized, cost is reduced, the rate of rehospitalization potentially is decreased, and patients ultimately become more confident and competent in caring for themselves. The healthcare team shifted its care philosophy to incorporate a care partner, increase patient control and independence, and create greater emphasis on education. Outcomes, including patient satisfaction, have demonstrated success and motivated expansion of this model to other patient populations.

Academic Medical Centers↗
PubMed1993

Phenotypic and functional characterization of competitive long-term repopulating hematopoietic stem cells enriched from 5-fluorouracil-treated murine marrow.

Lymphomyeloid stem cells from the bone marrow of C57BL/6 mice treated with 5-fluorouracil (5-FU) were characterized with respect to 12 parameters using fluorescence-activated cell sorting and a competitive long-term repopulation assay. Stem cells were larger than lymphocytes and exhibited side light-scatter characteristic of blast cells. Most expressed low levels of Thy-1.2, high levels of Sca-1 (Ly6-A/E), H-2Kb, and AA4.1 antigens and stained brightly with rhodamine-123. Significantly, most long-term repopulating cells also expressed CD4, some at high density. In addition, a significant proportion displayed low to medium levels of the "lineage-specific" markers CD45R (B220), Gr-1, and TER-119. A simple and rapid multiparameter sorting procedure enriched the stem cells 100-fold and substantially removed most other clonogenic cell types, including day 12 spleen colony-forming cells. Cells able to generate cobblestone colonies on stromal cells in vitro were co-enriched. Lethally irradiated mice transplanted with limiting numbers of the sorted stem cells did not survive unless cotransplanted with "compromised" marrow cells prepared by prior serial transplantation and shown to be depleted of long-term repopulating activity. A significant number of recipients transplanted with 25 to 100 sorted cells contained donor-derived B and T lymphocytes and granulocytes in their peripheral blood for at least 6 months. Limiting dilution analysis in vivo indicated that the frequency of competitive long-term repopulating units (CRU) in the sorted population was at least 1 in 60 cells. The calculated frequency of CRU was largely independent of the time of recipient analysis between 10 and 52 weeks, indicating that highly enriched stem cells can be recruited relatively early in certain transplant settings. This simple enrichment and assay strategy for repopulating hematopoietic stem cells should facilitate further analysis of their regulation in vivo.

Animals↗