Primitive hematopoietic stem cell function in vivo is uniquely high in the CXB-12 mouse strain

Bone marrow cells (BMCs) from CXB-12/HiaJ (CXB-12) mice had 14 times the total long-term repopulating ability found in the best of 11 other CXB recombinant inbred (RI) lines. BMCs from each RI line donor were mixed with genetically marked standard competitor BMCs from the BALB/cByxC57BL/6 F1 (CByB6F1) hybrid, the mice used to produce the RI lines, and the mixtures repopulated lethally irradiated CByB6F1 recipients. Percentages of donor-type erythrocytes and lymphocytes measured the actual long-term repopulating functions of the donor RI lines relative to the standard competitor. CXB-12 BMCs repopulated better after 3 or 6 months than after 1 month, suggesting that the most primitive precursors were involved. Compared to CByB6F1 standard competitor cells, CXB-12 cells repopulated 3 to 12 times as well, with their advantage increasing when higher doses of cells were transplanted, probably because of hybrid resistance of the recipient against low doses. This was far better than expected, because F1 cells normally function 2 to 3 times as well as cells from an inbred strain. In competitive dilution, the advantage resulted from 2 factors: more precursor cells and more function per precursor. In the model that best fit the data, CXB-12 donors had 2.4 times the concentration of hematopoietic stem cells (HSCs) as the CByB6F1 standard, and each HSC repopulated 1.4 times as well. CXB-12 mice did not have elevated erythrocyte and lymphocyte numbers in blood and marrow and did not have unusually elevated concentrations of colony-forming unit spleen, cobblestone colonies, and long-term colony-initiating cells in marrow.     

Effects of dietary restriction on hematopoietic stem-cell aging are genetically regulated

Diminished stem-cell functions with age may be a major cause of anemias and other defects. Unfortunately, treatments that increase stem-cell function can also increase the incidence of cancers. Lifelong dietary restriction (DR) is known to decrease spontaneous cancers and lengthen lifespan. This study examines the effect of DR on the ability of bone marrow cells to repopulate irradiated recipients and produce erythrocytes and lymphocytes. In BALB/cByJ (BALB) mice, repopulating abilities decline with age; DR ameliorates this trend. In C57BL/6J (B6) and (BALB x B6) F1 hybrid (F1) mice, repopulating abilities increase with age; DR maintains this increase. Hematopoietic stem cell (HSC) numbers are highly variable in aged BALB mice; however, the observed loss of marrow function results from a major loss in repopulating ability per HSC. DR greatly ameliorates this loss of function with age. In contrast, function per HSC in B6 mice is affected neither by age nor by DR. Thus, DR increases or maintains increased marrow repopulating ability with age in the 3 different genotypes tested, but effects on function per HSC depend on genotype. That DR increases or maintains stem-cell function with age, while decreasing cancer, has far-reaching health implications.     

A murine model of antimetabolite-based, submyeloablative conditioning for bone marrow transplantation: biologic insights and potential applications

OBJECTIVE: Nonmyeloablative conditioning regimens for marrow transplantation are desirable in many settings. Because repeated doses of the antimetabolite 5-fluorouracil (5-FU) decreases marrow long-term repopulating ability (LTRA) upon transplantation into lethally irradiated hosts, we hypothesized that mice given sequential doses of 5-FU (termed paired dose 5-FU) may permit substantial syngeneic marrow engraftment. METHODS: C57Bl/6 or X-linked chronic granulomatous disease (X-CGD) mice were administered 5-FU (150 mg/kg) on days -5 and -1. Assessment of host marrow phenotype and repopulating ability occurred on day 0. Transplantation of syngeneic donor marrow occurred on day 0 or day +15. RESULTS: We confirmed that the number of Sca-1+lin- cells and the LTRA of marrow from paired dose 5-FU-treated animals were diminished. C57Bl/6 hosts conditioned with paired doses of 5-FU followed by transplantation of 20 x 10(6) fresh B6.SJL marrow cells on day 0 displayed 44.9% +/- 7.1% donor chimerism 2 months posttransplant, and 34.4% +/- 8.6% donor chimerism 6 months posttransplant. In contrast, paired dose 5-FU-conditioned hosts transplanted with similar numbers of donor cells on day +15 exhibited only 3.4% +/- 1.2% donor chimerism at 2 months. Paired dose 5-FU-conditioned X-CGD hosts transplanted with MSCV-m91Neo-transduced X-CGD marrow averaged 6.6% +/- 2.3% (range, 4%-10%) NADPH oxidase-reconstituted neutrophils 12-16 months after transplant. CONCLUSION: These findings support the concept that impairment of host stem cell competitiveness may be an important mechanism for permitting engraftment of donor cells, and suggest that only a brief period of modest host stem cell impairment may be necessary to achieve substantial donor cell engraftment.     

Lymphohemopoietic reconstitution using wheat germ agglutinin-positive hemopoietic stem cell transplantation within but not across the major histocompatibility antigen barriers.

Nonadherent (NA), low density (LD), wheat germ agglutinin-positive (WGA+) murine hemopoietic stem cell-enriched preparations (HSCPs) were tested for the capability to reconstitute lymphohemopoietic elements in lethally irradiated mice. HSCPs from BALB/c mice reconstituted lethally irradiated, major histocompatibility complex (MHC)-matched DBA/2 mice to normal histology of the thymus and spleen and normal humoral and cellular immune functions. By contrast, lethally irradiated B6 mice could not be reconstituted after transplantation with NA, LD, WGA+ cells from MHC-mismatched BALB/c mice. We previously observed frequent survival, stable chimerism, and normally vigorous functioning immune systems in B6 mice transplanted with T-cell-depleted bone marrow from both BALB/c and B6 donors. To extend these findings to a stem cell transplantation system, lethally irradiated B6 mice were transplanted with NA, LD, WGA+ cells from both BALB/c and B6 mice. These mixed stem cell-enriched preparations did not reconstitute the lethally irradiated, MHC-mismatched mice. By contrast, such HSCPs from BALB/c plus DBA/2 into DBA/2 mice reconstituted the hematologic and lymphoid tissues and functional immune systems when the donor and the recipient pairs were matched at MHC and mismatched at multiminor histocompatibility barriers. These purified blood progenitors thus appear to lack certain cells/factors essential for engraftment and reconstituting recipients in a fully allogeneic environment.     

Marrow frequency of rat long-term repopulating cells: evidence that marrow hematopoietic stem cell concentration may be inversely proportional to species body weight

As measured by the long-term repopulating cell (LTRC) assay, only a few hematopoietic stem cells (HSCs) or perhaps a single HSC are required to totally repopulate the lymphohematopoietic tissues of lethally irradiated mice, cats, and humans, raising the question as to why large mammals require more marrow cells to either rescue them from lethal irradiation or establish a long-term hematopoietic graft than do small mammals. An explanation might be that HSC marrow frequency across species is not constant, but decreases as species body weight increases. This hypothesis was tested by comparing the LTRC marrow concentration of mice to that of rats. Specifically, histocompatible AKR/J Thy 1.1 marrow was transferred to 7-Gy irradiated C3H/HeN, Thy 1.2 mice, and histocompatible Norway Black marrow (NBr), RT 7.2 marrow was transferred to 7-Gy irradiated RT 7.1 Lewis rats. The recipients were scored for successful grafts 6 to 20 weeks later. By limiting dilution analysis, a value of 1 LTRC/47 700 marrow cells was calculated for mice, but only 1 LTRC/502,000 marrow cells was calculated for rats. Viewed in the context of marrow grafting in larger mammals, these results suggest that species with greater body mass have lower marrow HSC frequency.     

Transplantation of Autoimmune Potential. I. Development of Antinuclear Antibodies in H-2 Histocompatible Recipients of Bone Marrow from New Zealand Black Mice

Antinuclear autoantibodies (ANA) appeared in the plasma of lethally irradiated H-2d histocompatible DBA/2 and BALB/c mice several weeks after intravenous transplantation of 2 to 4 x 10(6) bone marrow cells from 3-week-old animals of the autoimmune New Zealand Black (NZB) strain. Little or no ANA development was observed in DBA/2 or BALB/c strains when syngeneic or nonautoimmune allogeneic marrow was grafted, or when NZB marrow was injected into untreated DBA mice or mice receiving 200 rads of x-irradiation. Transfer of 5 x 10(6) spleen cells from 8-day-old NZB mice into lethally irradiated BALB/c mice effected substantial ANA formation by the ninth day after transfer, compared to a 20-day latency following transfer of the same number of bone marrow cells. This earlier conversion with splenocytes may have been due to the presence of immunocompetent T and B cells, since stem cell numbers of the two tissues were similar. Transplantation of NZB marrow to lethally irradiated H-2 incompatible SJL/J (H-2s) and C57B1/6 (H-2b) strains brought about less ANA conversion than the transfer of compatible (SJL x NZB)F1 and (C57B1 x NZB)F1 marrow cells to the respective nonautoimmune SJL or C57B1 parental strain. Graft-versus-host reactions thus did not appear to play a requisite or determining role in the autoimmune development observed following grafting of NZB hemopoietic tissues. Reconstitution of lethally irradiated NZB mice with BALB/c or SJL/J bone marrow depressed the recurrence of ANA for 30 days, compared to rapid ANA recovery following NZB marrow injection. The characteristics that ultimately provoke or permit spontaneous auto-reactivity are inherent in the hemopoietic stem cell population of the NZB strain.     

Genetic regulation of primitive hematopoietic stem cell senescence

OBJECTIVE: To define effects of strain on PHSC (primitive hematopoietic stem cells) senescence (decline in function with age) in vivo, and to map a locus that regulates PHSC senescence. MATERIALS AND METHODS: Long-term function and self-renewal were compared in bone marrow cells (BMC) from old and young mice of three strains: BALB/cBy (BALB), DBA/2 (D2) and C57BL/6 (B6), using competitive repopulation and serial transplantation in vivo. BMC from each old or young donor were mixed with standard doses of congenic, genetically marked BMC and transplanted into lethally recipients. Percentages of donor-type erythrocytes and lymphocytes in the recipients determined the functional ability of donor PHSC relative to the standard, where one repopulating unit (RU) of donor BMC equals the repopulating ability of 100,000 standard competitor BMC. Using similar techniques, repopulating abilities of old and young recombinant inbred (RI) donors of 12 strains derived from BALB and B6 were compared in NK-depleted BALBxB6 Fl recipients to map a locus that appears to have a major role in PHSC senescence. RESULTS: PHSC function declined about 2 fold with age in BALB and D2 BMC, and increased more than 2-fold with age in B6 BMC, with all old/young strain differences significant, p<.01. Ten months after serial transplantation, young B6, BALB, and D2 PHSC had self-renewed 1.6-, 4.2-, and 3.2-fold better than old, with BALB and D2 old/young differences p<.01. Young B6 PHSC self-renewed 1.9- and 2.9-fold better than young BALB and D2 PHSC. The PHSC senescence phenotypes (old/young RU ratios) for 12 CXB RI strains suggested a genetic linkage to D12Nyul7 on Chromosome 12. CONCLUSION: PHSC senescence is genetically regulated, and is much delayed in the B6 strain compared to the BALB and D2 strains. A locus on Chromosome 12 may regulate PHSC senescence.     

Low level of gene transfer to and engraftment of murine bone marrow cells from long-term bone marrow cultures

OBJECTIVE: We wanted to determine whether the long-term bone marrow culture (LTBMC) transduction system would lead to efficient gene transfer and engraftment of murine repopulating hematopoietic stem cells (HSC), particularly in nonablated recipients. MATERIALS AND METHODS: Congenic mouse strains expressing Ly 5.1 or Ly 5.2 and the GP+E86 cell line producing the MGirL22Y vector carrying the gene for enhanced GFP were used. Murine LTBMCs were established and demi-depopulated on days 7 and 14 with addition of vector supernatant on days 8 and 15. RESULTS: Cell recovery on day 21 was 21.3%+/-3.8% of input cells and CFU-C recovery was 9.7+/-3.4% as compared with CFU-C of input cells. In vitro transduction efficiency determined by CFU-C expressing GFP was 22.2%+/-1.6%. In irradiated (950 cGy) mice transplanted with 2x10(6) LTBMC cells, 94% of nucleated cells in the blood at week 16 were of donor origin. However, GFP was only detected at low level in a few animals at week 4 and not later. Analysis of bone marrow from these mice at week 20 did not show any GFP expression and semiquantitative PCR revealed a transgene level of <1%. When 3.5-20.8x10(6) LTBMC cells (corresponding to 20-100x10(6) fresh cells) were transplanted to nonablated recipients, no engraftment or GFP expression were detected. Competitive repopulation experiments showed that the long-term repopulation ability (LTRA) of the LTMC cells was only 7% of fresh cells. CONCLUSION: These results indicate that LTBMC transduction of murine cells leads to low-level transduction of progenitors, no gene transfer to repopulating stem cells, and reduction in LTRA in ablated and nonablated recipients.     

Marrow graft rejection by repeated transfusions of allogeneic donor spleen cells

Many hematological diseases require long-term transfusion support, which causes production of donor-reactive antibodies in sensitized recipients. Sensitized patients are at an increased risk for graft rejection when they undergo allogeneic hematopoietic stem cell transplantation (allo-HSCT). Here, we established a highly sensitized murine model to investigate the mechanism of donor graft rejection. After BALB/c mice were repeatedly transfused with allogeneic spleen cells from C57BL/6 mice, there was a significant increase in complement-dependent cytotoxicity in the serum of sensitized mice. For transplantation, 1 x 10(7) bone marrow cells (BMCs) from C57BL/6 mice were injected into lethally irradiated recipient BALB/c mice. Sensitized mice died between 12 and 15 days post-transplantation, while non-sensitized mice remained alive after 28 days. The hematopoietic recovery rate declined over time in sensitized recipients. The homing trace assay showed a rapid disappearance of donor BMCs in the spleen and bone marrow of sensitized recipients. In addition, the recipient cells and antibodies in the sensitized serum were capable of inducing high level of cell- and complement-mediated cytotoxicity to the donor graft. Our finding may explain the impaired hematopoietic stem cell homing and poor hematopoietic engraftment observed in highly sensitized allo-HSCT patients.     

Ex vivo culture rescues hematopoietic stem cells with long-term repopulating capacity following harvest from lethally irradiated mice

OBJECTIVE: High-dose ionizing radiation can cause lethal myeloablation in exposed individuals. We examined whether ex vivo culture could rescue hematopoietic stem cells with repopulating capacity following harvest from lethally irradiated animals. METHODS: We exposed B6.SJL mice to 1050 cGy, harvested their irradiated bone marrow (BM), and examined whether ex vivo culture of the irradiated BM mononuclear cells (MNC) with porcine microvascular endothelial cells (PMVEC) or cytokines alone could rescue hematopoietic cells with in vitro colony-forming activity, in vivo radioprotective capacity, and long-term repopulating potential. RESULTS: PMVEC coculture supported the recovery of fourfold and 80-fold greater numbers of total cells and colony-forming cells (CFC) compared to cyokines alone following 1050 cGy irradiation. All control mice irradiated with 1050 cGy died by day 30, as did mice transplanted with 1050 cGy-irradiated BM MNC. In contrast, transplantation of 1050 cGy-irradiated/PMVEC-cultured BM was fully radioprotective in 12 of 16 recipient mice (75%) exposed to 1050 cGy. Six of the 12 CD45.2+ mice (50%) transplanted with 1050 cGy-irradiated/PMVEC-cultured cells showed long-term (>6 months) multilineage repopulation derived from irradiated donor CD45.1+ cells. Surprisingly, transplantation of identical doses of 1050 cGy-irradiated/cytokine-cultured BM was also radioprotective in 50% of irradiated recipient mice and 50% of these mice demonstrated donor-derived repopulation. CONCLUSIONS: Fully functional BM stem and progenitor cells can be rescued following harvest from lethally irradiated animals via ex vivo culture with PMVEC or cytokines alone. This method can serve as a model for the rapid ex vivo rescue and transplantation of autologous BM progenitors in the treatment of victims of radiation injury.     

The decrease in long-term marrow repopulating capacity seen after transplantation is not the result of irradiation-induced stromal injury

Marrow cells from nonirradiated F1-W/Wv mice repopulated slightly less well than cells from lethally irradiated recipients. Therefore, avoiding irradiation of recipients did not improve the relative repopulating ability of their marrow cells. In other experiments, F1-W/Wv mice were transplanted by parabiosis with marrow of WBB6F1-+/+ (F1-+/+) mice, avoiding cellular handling and irradiation. Marrow cells transplanted to F1-W/Wv mice by this procedure demonstrated slightly better repopulating ability than did marrow cells transplanted by injection. However, they performed no better than those transplanted by parabiosis to irradiated F1-+/+ recipients. Significant impairment of stromal function after irradiation was not indicated. Apparently, stem cell damage caused by transplantation may have greater importance in causing loss of stem cell replicative potential than effects of irradiation-induced stromal injury.     

Autonomous behavior of hematopoietic stem cells

Mechanisms that affect the function of primitive hematopoietic stem cells with long-term proliferative potential remain largely unknown. Here we assessed whether properties of stem cells are cell-extrinsically or cell-autonomously regulated.We developed a model in which two genetically and phenotypically distinct stem cell populations coexist in a single animal. Chimeric mice were produced by transplanting irradiated B6D2F1 (BDF1) recipients with mixtures of DBA/2 (D2) and C57BL/6 (B6) day-14 fetal liver cells.We determined the mobilization potential, proliferation, and frequency of D2 and B6 stem and progenitor cells in animals with chimeric hematopoiesis. After granulocyte colony-stimulating factor (G-CSF) administration, peripheral blood D2 colony-forming units granulocyte-macrophage were fourfold to eightfold more numerous than B6 progenitors. We determined that D2 and B6 progenitors maintained their genotype-specific cycling activity in BDF1 recipients. Chimeric marrow was harvested and D2 and B6 cell populations were separated by flow cytometry. Cobblestone area-forming cell (CAFC) analysis of sorted marrow showed that the number of late appearing CAFC subsets within the D2 cell population was approximately threefold higher than within the B6 fraction. We performed secondary transplantation using unfractionated chimeric marrow, which was given in limiting doses to lethally irradiated BDF1 recipients. Comparison of the proportion of animals possessing D2 and/or B6 leukocytes 5 months after transplant revealed that the frequency of D2 LTRA was approximately 10-fold higher than B6 LTRA numbers.Our data demonstrate that genetically distinct stem cell populations, coexisting in individual animals, independently maintain their parental phenotypes, indicating that stem cell properties are predominantly regulated cell-autonomously.     

Hemopoietic stem cell transplantation using mouse bone marrow and spleen cells fractionated by lectins.

Mouse bone marrow and spleen cells were fractionated with the aid of soybean agglutinin and peanut agglutinin. A test for spleen colony-forming units in the isolated fractions showed that the hemopoietic stem cells are agglutinated by both of these lectins. The capacity of the agglutinated fractions to reconstitute lethally irradiated allogeneic mice was investigated. A sequential fractionation of splenocytes from SWR donors by soybean agglutinin and peanut agglutinin, or a single fractionation by soybean agglutinin of splenocytes from BALB/c donors, afforded a cell fraction that successfully reconstituted lethally irradiated (BALB/c X C57BL/6)F1 mice, without complications due to graft-versus-host reaction.     

In vitro transdifferentiation of adult bone marrow Sca-1+ cKit- cells cocultured with fetal liver cells into hepatic-like cells without fusion

Several research groups have recently reported that certain bone marrow cells (BMCs) differentiate into hepatocytes in vitro as well as in vivo in rodents. However, it has yet to be elucidated what factors effectively trigger and sustain transdifferentiation of BMCs. In the present study, we specifically asked whether the presence of murine fetal liver cells (FLCs) triggered and supported in vitro transdifferentiation of murine BMCs. Fractionated BMCs from green fluorescence protein (GFP)-expressing transgenic mice and FLCs from ROSA26 mice (X-gal(+) FLCs) were cocultured in the presence of hepatocyte growth factor in laminin-coated dishes. We found that Sca-1(+) BMCs gave rise to adherent hepatic-like cells, which expressed albumin as assessed with immunocytochemistry and RNA-polymerase chain reaction (PCR), and alpha-fetoprotein and cytokeratin 19 as examined with RNA-PCR. When GFP(+)Sca-1(+)cKit(-) cells were cocultured with X-gal(+) FLCs, all GFP(+) albumin-producing cells were negative for X-gal, showing that cell fusion was not associated in the observed BMCs' differentiation into hepatic-like cells. Titration analysis revealed that 1 of 5,943 Sca-1(+)cKit(-) cells had the ability to proliferate and differentiate into hepatic-like cells. These data strongly suggest that BMCs differentiate into hepatic-like cells in the presence of FLCs and that the present method may be useful for propagating BMC-derived hepatocytic progenitors and for investigating the nature of those cells.     

Myelosuppressive conditioning is required to achieve engraftment of pluripotent stem cells contained in moderate doses of syngeneic bone marrow

We have investigated the requirement for whole body irradiation (WBI) to achieve engraftment of syngeneic pluripotent hematopoietic stem cells (HSCs). Recipient B6 (H-2b; Ly-5.2) mice received various doses of WBI (0 to 3.0 Gy) and were reconstituted with 1.5 x 10(7) T-cell- depleted (TCD) bone marrow cells (BMCs) from congenic Ly-5.1 donors. Using anti-Ly-5.1 and anti-Ly-5.2 monoclonal antibodies and flow cytometry, the origins of lymphoid and myeloid cells reconstituting the animals were observed over time. Chimerism was at least initially detectable in all groups. However, between 1.5 and 3 Gy WBI was the minimum irradiation dose required to permit induction of long-term (at least 30 weeks), multilineage mixed chimerism in 100% of recipient mice. In these mice, stable reconstitution with approximately 70% to 90% donor-type lymphocytes, granulocytes, and monocytes was observed, suggesting that pluripotent HSC engraftment was achieved. About 50% of animals conditioned with 1.5 Gy WBI showed evidence for donor pluripotent HSC engraftment. Although low levels of chimerism were detected in untreated and 0.5-Gy-irradiated recipients in the early post-BM transplantation (BMT) period, donor cells disappeared completely by 12 to 20 weeks post-BMT. BM colony assays and adoptive transfers into secondary lethally irradiated recipients confirmed the absence of donor progenitors and HSCs, respectively, in the marrow of animals originally conditioned with only 0.5 Gy WBI. These results suggest that syngeneic pluripotent HSCs cannot readily engraft unless host HSCs sustain a significant level of injury, as is induced by 1.5 to 3.0 Gy WBI. We also attempted to determine the duration of the permissive period for syngeneic marrow engraftment in animals conditioned with 3 Gy WBI. Stable multilineage chimerism was uniformly established in 3-Gy-irradiated Ly-5.2 mice only when Ly-5.1 BMC were injected within 7 days of irradiation, suggesting that repair of damaged host stem cells or loss of factors stimulating engraftment may prevent syngeneic marrow engraftment after day 7.     

Proliferation of totipotent hematopoietic stem cells in vitro with retention of long-term competitive in vivo reconstituting ability.

Marrow cells from male mice pretreated with 5-fluorouracil were infected with helper-free neomycin-resistant (neor) recombinant retrovirus and then used to initiate long-term cultures (LTC) on irradiated adherent marrow feeder layers. Four weeks later LTC cells were harvested and injected into lethally irradiated female recipients either alone or together with 2 x 10(5) female marrow cells with selectively compromised long-term repopulating potential to assay for totipotent and competitive repopulating units (CRU), respectively. A total of 46 unique clones were detected in recipients 5 wk to 7 mo after transplant. Half of these clones (22 of 46) included both lymphoid and myeloid progeny. Eight of the 22 lympho-myeloid clones were represented in multiple recipients, in some cases after injection of limiting numbers of CRU, thus indicating repopulation from sibling totipotent stem cells generated during the initial 4-wk period in LTC. Serial analysis of cells released into the nonadherent fraction of LTC for up to 7 wk provided additional evidence of the continuing proliferation in LTC of totipotent stem cells with long-term repopulating potential. The frequency of CRU determined from limiting-dilution analyses of LTC-derived cells was the same for recipients analyzed at 5 wk or 7 mo after transplantation and was also the same whether marrow or thymus repopulation was assessed. These assays showed that concurrent with the expansion of some totipotent cells revealed by retroviral marking, there was a slow but net 6.5-fold decrease in total CRU numbers after 4 wk in LTC. These results show the capacity of some totipotent hematopoietic stem cells to be maintained and amplified over extensive time periods in vitro without diminution of their long-term in vivo repopulating potential. These results also set the stage for analogous studies of human stem cell selection and expansion in vitro, which may be important for future gene therapy protocols.     

Hematopoietic stem cell tracking in vivo: a comparison of short-term and long-term repopulating cells

We have previously demonstrated that we could separate long-term repopulating stem cells from cells that provided radioprotection (short-term repopulating cells) on the basis of size and suggested that this might be due to the quiescent nature of long-term repopulating cells. To further define the activity of these populations, we used a dye (PKH26), which incorporates into the membrane of cells and is equally distributed to daughter cells when they divide. We developed an assay, which allowed us to retrieve PKH26(+) long-term and short-term repopulating cells in the hematopoietic tissues of the recipients posttransplant. We were able to recover the labeled cells and determine their cell cycle activity, as well as their ability to reconstitute secondary lethally irradiated hosts in limiting dilution. The results of our assay suggest that long-term repopulating cells are quiescent in the bone marrow (BM) 48 hours after transplant. We were able to detect only a few labeled cells in the peripheral blood posttransplant and even though cells homed to both the spleen and BM, more long-term repopulating cells homed to the marrow and only these cells, which homed to the marrow, were capable of reconstituting lethally irradiated secondary hosts long-term.     

Mesenteric hemopoietic colonies: occurrence in BALB/c mice after transplantation of syngeneic normal or leukemic hemopoietic cells.

Intraperitoneal (ip) inoculation of BALB/c mice with syngeneic hemopoietic cells results in the formation of 'Mesenteric Hemopoietic Colonies' (MHC). In lethally irradiated mice actively growing erythroid, myeloid and megakaryocytic, or mixed colonies form and soon become confluent. It is therefore concluded that in mice the mesentery is a suitable site for growth of hemopoietic cells. The mesentery might play an important role in the recovery of the hemopoietic system in lethally irradiated mice, being the primary site of proliferation of stem cells and/or CFU before their migration to bone marrow and spleen. Bone marrow and spleen cells from animals infected with Rauscher Leukemia Virus (R-MuLV) also produce MHC and spleen colonies after ip injection into lethally irradiated mice. In addition to the undifferentiated cells in the MHC, cells with limited differentiation and/or retarded maturation were identified. The cytologic pattern of the majority of cells in MHC was of mixed type.     

Expansion of hematopoietic stem cell phenotype and activity in Trp53-null mice

OBJECTIVES: To study the effects of transformation-related protein 53 (Trp53) and other genes on hematopoiesis and hematopoietic stem cells (HSCs). METHODS: Frequencies of murine bone marrow cells (BMCs) with the Lin(-)Sca-1(+)c-kit(+)CD34- phenotype were analyzed by flow cytometry, and were increased in mice with germ-line deletion of the Trp53 (Trp53(-/-)) gene but not in 25 other deletions of genes involved in cell cycling, development, cancer, or hematopoiesis. Therefore, Trp53(-/-) and wild-type Trp53(+/+) mice were compared using the following assays: complete blood counts, day-9 colony-forming unit spleen (CFU-S), and competitive repopulation. In the latter assay, donor repopulating ability was analyzed at one, three, and five months, while recipient survival and recipient blood and bone marrow cell composition were analyzed at five months, after transplantation. RESULTS: In comparison to wild-type controls, Trp53(-/-) mice had normal blood and bone marrow cell counts, increased CD11b(+), and decreased CD45R(+) cell proportions in blood and bone marrow, twice as many Lin(-)Sca-1(+)c-kit(+)CD34(-) BMCs, and 37% more day-9 CFU-S. In the competitive repopulation assay, Trp53(-/-) BMCs engrafted lethally irradiated recipients two to four times better than Trp53(+/+) BMCs. The Trp53(-/-) engraftment advantage increased with time in the recipients. Recipients of Trp53(-/-) donors had two to three times more Lin(-)Sca-1(+)c-kit(+)CD34(-) BMCs than recipients of Trp53(+/+) donors at five months after transplantation. However, only 44% of recipients of Trp53(-/-) donors survived five months after trans-plantation, compared with 92% of recipients of Trp53(+/+) donors. CONCLUSION: The Trp53-null allele expands bone marrow Lin(-)Sca-1(+)c-kit(+)CD34(-) cells and the overall activity of HSCs; however, it increases recipient mortality.