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Results for “stem cell competition”

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PubMed2005

Competitive engraftment of hematopoietic stem cells genetically modified with a truncated erythropoietin receptor.

Transplantation of genetically modified hematopoietic stem cells (HSCs) has therapeutic potential for a variety of blood genetic disorders. Engraftment of HSCs, however, requires toxic myeloablative treatments, which render this approach questionable for non-life-threatening disorders. A potential alternative is the use of transgenes, which allows positive selection of HSCs in vivo. We used retroviral vectors to express a truncated derivative of the erythropoietin receptor (tEpoR) in murine and human hematopoietic cells. Murine HSCs expressing tEpoR at different levels (1500 to 13,000 receptors/cell) acquire a competitive repopulation capacity in vivo upon transplantation into fully or partially myeloablated co-isogenic mouse recipients. Long-term analysis of transplanted mice showed that expression of tEpoR at paraphysiological levels (approximately 1500 receptors/cell) has no effect on steady-state hematopoiesis and induces no further expansion of transduced cells after the engraftment period. Human cord blood-derived CD34+ stem/progenitor cells transduced with a lentiviral vector expressing tEpoR expand their clonogenic capacity in vitro, and significantly increase their marrow repopulation capacity upon xenotransplantation into sublethally irradiated NOD-SCID mice, with no alteration in their phenotype, survival, and differentiation properties. These data indicate that expression of tEpoR is an effective strategy to promote selective engraftment of genetically modified HSCs upon transplantation in both myeloablative and nonmyeloablative conditions, without the use of toxic drugs for selection.

Animals↗

PubMed2002

A novel dynamic model of hematopoietic stem cell organization based on the concept of within-tissue plasticity.

OBJECTIVE: At present, no dynamic quantitative models of stem cell organization are available that fulfill all criteria of the prevalent functional definition of hematopoietic stem cells and, at the same time, provide a consistent explanation of cell kinetic and functional stem cell heterogeneity, reversibility of cellular properties, self-organized regeneration after damage, fluctuating activity and competition of stem cell clones, and microenvironment dependency of stem cell quality. To solve this problem, we propose a new, comprehensive model concept. MATERIALS AND METHODS: A single cell-based stochastic model is described. It makes the novel concept of within-tissue plasticity operational. Within a range of potential options, individual cells may reversibly change their actual set of properties depending on the influence of the local growth environment. Stochastic switching between the growth environments introduces fluctuations that eventually generate heterogeneity. Extensive model simulations are compared with experimental data. RESULTS: Although stemness is not an explicit cellular model property, the system behavior is consistent with the functional definition of stem cells and explains a large set of experimental observations on stem cell function in vivo and in vitro on the level of cell populations and individual cells. Classic results such as the colony-forming unit spleen assay, as well as recent experimental observations on stem cell kinetics, individual clone tracking, and fluctuating clonal contribution, are discussed. CONCLUSIONS: This concept introduces a fundamentally new perspective on stem cell organization treating stemness not as an explicit cellular property but as the result of a dynamic process of self-organization. The model needs to be extended to incorporate lineage specification and tissue plasticity.

Animals↗

PubMed2006

Long-term engraftment of p18(INK4C)-deficient hematopoietic stem cells is enhanced in the sublethally-irradiated recipients.

Non-myeloablative regimens for host conditioning have been widely used in clinical hematopoietic stem cell transplantation due to their reduced toxicity on the recipients. But a milder conditioning regimen may require a higher engrafting ability of donor stem cells in competing with endogenous stem cells. Thus, new strategies for enhancing the competitiveness of donor stem cells in non-myeloablative recipients would have important implications for current clinical stem cell transplantation. It is known that the absence of p18(INK4C) (p18) gene can enhance the self-renewal potential of hematopoietic stem cells (HSCs). We applied the approach of competitive bone marrow transplantation to evaluate the impact of p18 gene deletion on long-term engraftment of HSCs in sublethally irradiated hosts. We found that p18(-/-) HSCs had a significant advantage over wild-type HSCs during long-term engraftment in the mouse recipients that received a sub-lethal irradiation (5-Gy). The engraftment efficiency of p18(-/-) HSCs in the sub-lethally irradiated recipients was similar to that in the lethally irradiated (10-Gy) recipients. Our current study demonstrates that enhanced engraftment of donor HSCs in the absence of p18 does not strictly depend on the dose of irradiation used for host conditioning. Therefore, p18 might serve as a potential drug target for increasing the efficacy of stem cell transplant in the patients that are preconditioned with either a myeloablative or non-myeloablative regimen.

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PubMed2006

Fibroblast growth factor-1 and -2 preserve long-term repopulating ability of hematopoietic stem cells in serum-free cultures.

In this study, we demonstrate that extended culture of unfractionated mouse bone marrow (BM) cells, in serum-free medium, supplemented only with fibroblast growth factor (FGF)-1, FGF-2, or FGF-1 +2 preserves long-term repopulating hematopoietic stem cells (HSCs). Using competitive repopulation assays, high levels of stem cell activity were detectable at 1, 3, and 5 weeks after initiation of culture. FGFs as single growth factors failed to support cultures of highly purified Lin(-)Sca-1(+)c-Kit(+)(LSK) cells. However, cocultures of purified CD45.1 LSK cells with whole BM CD45.2 cells provided high levels of CD45.1 chimerism after transplant, showing that HSC activity originated from LSK cells. Subsequently, we tested the reconstituting potential of cells cultured in FGF-1 + 2 with the addition of early acting stimulatory molecules, stem cell factor +interleukin-11 + Flt3 ligand. The addition of these growth factors resulted in a strong mitogenic response, inducing rapid differentiation and thereby completely overriding FGF-dependent stem cell conservation. Importantly, although HSC activity is typically rapidly lost after short-term culture in vitro, our current protocol allows us to sustain stem cell repopulation potential for periods up to 5 weeks.

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PubMed1993

Assessing permanent damage to primitive hematopoietic stem cells after chemotherapy using the competitive repopulation assay.

The competitive repopulation assay was used to document the effects of six chemotherapeutic agents on primitive hematopoietic stem cells. The assay measures the relative abilities of donor cells to produce circulating erythrocytes and lymphocytes in lethally irradiated congeneic mice over a period of 6 months. Long-lasting marrow reconstitutive deficits in cells of donor origin occurred after exposure to 5-fluorouracil (5FU), bis-chloronitrosourea (BCNU), cyclophosphamide (CTX), vincristine (VCR), and actinomycin D (ACT) but not after exposure to cytosine arabinoside (ARA). Repopulating abilities were reduced after as little as a single dose of CTX or BCNU. A second dose of BCNU caused even more severe effects. A single dose of 5FU had no effect on repopulating abilities despite a temporary 10-fold reduction in marrow cell number, but multiple doses reduced the marrow stem-cell replicative ability to less than half of the normal control levels. These effects were not reliably predicted or detected by colony-forming assays or by reductions in marrow cell number. Thus, long-lasting proliferative defects in the primitive hematopoietic stem-cell (PHSC) population can result from the use of chemotherapeutic agents. Such findings may have clinical implications, especially in individuals receiving repeated or prolonged administration of these agents or in instances of marrow transplantation.

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PubMed2005

Genetic reduction of class IA PI-3 kinase activity alters fetal hematopoiesis and competitive repopulating ability of hematopoietic stem cells in vivo.

Class I(A) phosphatidylinositol-3 kinase (PI-3K) is a lipid kinase, which is activated in blood cells by hematopoietic growth factors. In vitro experiments using chemical inhibitors of PI-3K suggest that this kinase is potentially important for hematopoietic stem and progenitor cell (HSC/P) function, and recent studies identify PI-3K as a therapeutic target in treating different leukemias and lymphomas. However, the role of PI-3K in regulating fetal liver or adult hematopoiesis in vivo is unknown. Therefore, we examined PI-3K-deficient embryos generated by a targeted deletion of the p85alpha and p85beta regulatory subunits of PI-3K (p85alpha-/-p85beta+/-). The absolute frequency and number of hematopoietic progenitor cells were reduced in p85alpha-/- p85beta+/- fetal livers compared with wild-type (WT) controls. Further, p85alpha-/-p85beta+/- fetal liver hematopoietic stem cells (HSCs) had decreased multilineage repopulating ability in vivo compared with WT controls in competitive repopulation assays. Finally, purified p85alpha-/-p85beta+/- c-kit+ cells had a decrease in proliferation in response to kit ligand (kitL), a growth factor important for controlling HSC function in vivo. Collectively, these data identify PI-3K as an important regulator of HSC function and potential therapeutic target in treating leukemic stem cells.

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PubMed1998

Equal distribution of competitive long-term repopulating stem cells in the CD34+ and CD34- fractions of Thy-1lowLin-/lowSca-1+ bone marrow cells.

CD34 antigen is present on most, if not all, human hematopoietic stem cells (HSCs). Consistent with this pattern of expression, we recently reported that primitive murine HSCs defined as competitive long-term repopulating units (CRUs) are highly enriched among CD34+ bone marrow (BM) cells (one CRU/2500 cells). However, in agreement with one recent report that some murine HSCs do not express CD34 (Science 273:242), we observed that 15% of phenotypically defined Thy-1lowLin-/lowSca-1+ (TLS) stem cells were CD34- by fluorescence-activated cell sorting. To examine further the nature of CD34 expression on murine hematopoietic cells, we separated TLS cells into CD34+ (0.022% of BM cells) and CD34- (0.005% of BM cells) fractions, confirmed their phenotype by reverse transcriptase-polymerase chain reaction analysis of CD34 transcripts, and evaluated them in a variety of in vitro and in vivo assays. The CD34+ TLS population contained most (93-95%) of the day 12 spleen colony-forming units (CFU-S) and in vitro colony-forming cells (CFCs). Cobblestone area-forming cells (CAFCs) able to proliferate on a murine bone marrow stromal cell line (SyS-1) represented one of every 5 CD34+ TLS and one of every 31 CD34 TLS cells. When lethally irradiated mice were injected with 100 CD34+ TLS cells, all animals survived and began to recover circulating leukocytes, platelets, and erythrocytes by 15 days. In contrast, only 40% of mice injected with 100 CD34- TLS cells were radioprotected, and hematopoietic reconstitution in surviving mice was not apparent until 21 days. The frequency of CRUs in CD34+ and CD34 TLS cells was determined by injecting limiting numbers of cells into lethally irradiated Ly-5 congenic hosts together with 10(5) "compromised" BM cells to provide radioprotection. CRUs able to regenerate and maintain lymphoid and myeloid cells for at least 6 months in primary and 5 months in secondary hosts represented one of every 156 CD34+ TLS and one of every 35 CD34- TLS cells. However, when normalized for the proportion of TLS cells that were CD34+ or CD34-, it was determined that the recovery of CRU among CD34+ and CD34- TLS cells was equivalent (46% and 54%, respectively). These data are consistent with the previous description of repopulating HSCs among CD34-c-kit+Sca-1+Lin- cells (Science 273:242, 1996) and provide additional evidence that TLS cells are functionally heterogeneous and can be further fractionated on the basis of CD34 expression. Overall, approximately 95% of CFCs, CFU-S, and CAFCs in the TLS population were found to be CD34+, whereas more primitive CRU were distributed equally among CD34+ and CD34 TLS cells. These results should enable better characterization of the most primitive stem cells in murine BM.

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PubMed1997

Early transplantation to a normal microenvironment prevents the development of Steel hematopoietic stem cell defects.

Our previous results showed that hematopoietic stem cells from 16-week-old Sl/Sl(d) mice are not as competitive as congenic +/+ control stem cells. Possible explanations for these findings are that the Steel stem cells are either inherently defective or lose competitive ability by residence in an environment lacking membrane-bound Steel factor. In the present report, any long-term effects of the Steel microenvironment were eradicated by transferring neonatal Sl(d)/Sl(d) marrow and spleen cells into an irradiated but otherwise normal adult hematopoietic microenvironment. Host cells were completely replaced by donor cells within 6 weeks. Eight months after transplantation, the Sl(d)/Sl(d) and similarly treated +/+ littermate control cells from the primary recipient marrow were competed against genetically marked normal cells in an irradiated secondary host. The Steel cells were as competitive as the control cells demonstrating that Steel stem cells are not inherently defective. Results suggest that the stem cells, when retained in the mutant environs into adulthood, are either reduced in number or phenotypically altered by lack of the membrane-bound Steel factor.

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PubMed2005

Characterization of the human CD34+ hematopoietic progenitor cell compartment during the second trimester of pregnancy.

BACKGROUND AND OBJECTIVES: Characterization of the different sites of fetal hematopoiesis during the second trimester of pregnancy can provide important information for the timing of in utero stem cell transplantation (SCT), as an experimental treatment for congenital hematologic disorders. DESIGN AND METHODS: We analyzed the distribution of the different hematopoietic precursor cells in fetal blood, liver, bone marrow (BM), spleen and thymus from 66 fetuses between the ages of 13 to 23 weeks of gestation by flow cytometry and culture of hematopoietic progenitor cells (HPC) in semi-solid media. RESULTS: During the second trimester the percentages of CD34+ cells did not change and were 4.0% (1.0-12.0%) (median [min.-max.]) in blood, 16.5% (3.0-32.0%) in BM, 6.0% (2.0-16.0%) in liver, 5.0% (2.0-14.0%) in spleen, and 1.1% (0.9-3.0%) in the thymus. Each tissue contained all subsets of CD34+ cells at various levels. Within the CD34+ population, in BM the main sub-population was CD34+CD19+ (38% (11-67%)), in thymus CD34+ CD7+ (83% [45-98%]), and in blood and liver CD34+ CD33+ (57% (30-80%) and 48% (20-82%), respectively). In all tissues approximately 1 % of nucleated cells were non-committed CD34+ CD38- cells. The frequencies of both committed CD34+ cells and non-committed CD34+ CD38- cells were constant from 13 to 23 weeks in fetal blood, BM, liver and spleen. The frequencies of cultured HPC were high in fetal liver, low in fetal BM, and increasing in fetal blood. INTERPRETATION AND CONCLUSIONS: During the second trimester of gestation, all CD34+ subsets were present in each hematopoietic compartment at different levels. An exchange of stem cells between organs is likely, but no major shift of the hematopoietic stem cell compartment from the liver to other hematopoietic organs was found during the mid-trimester. No arguments for a specific time window for performing in utero SCT were found, but if engraftment of donor stem cells in the human fetus is influenced by competition of endogenous stem cells or fetal immune competence, in utero SCT should be performed as early as possible during fetal development.

Antigens, CD19↗

PubMed2006

Fibronectin and laminin enhance engraftibility of cultured hematopoietic stem cells.

To test the hypothesis that extracellular matrix (ECM) components maintain stem cell property, murine bone marrow (BM) cells were expanded in fibronectin and laminin coated plate in the presence of cytokines. We observed significant phenotypic and functional improvement of expanded cells. In 10 days, 800-fold expansion of colony-forming unit-granulocyte erythrocyte monocyte megakaryocyte (CFU-GEMM) was observed in the cultured cells. No apparent activation of cell cycle was observed, but CD29 and very late antigen-4 (VLA-4) expression was increased, as compared to the normal BM cells. A fraction of the expanded cells became verapamil sensitive, suggesting upregulation of multi-drug resistant gene(s), as found in the primitive hematopoietic stem cells (HSCs). Competitive repopulation assay confirmed that HSCs compartment was amplified during culture. Overall, our study clearly demonstrated that ex vivo culture of murine HSCs in the presence of fibronectin and laminin resulted in expansion of primitive stem cells and improvement in the marrow engraftibility.

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PubMed2005

APETALA2 regulates the stem cell niche in the Arabidopsis shoot meristem.

Postembryonic organ formation in higher plants relies on the activity of stem cell niches in shoot and root meristems where differentiation of the resident cells is repressed by signals from surrounding cells. We searched for mutations affecting stem cell maintenance and isolated the semidominant l28 mutant, which displays premature termination of the shoot meristem and differentiation of the stem cells. Allele competition experiments suggest that l28 is a dominant-negative allele of the APETALA2 (AP2) gene, which previously has been implicated in floral patterning and seed development. Expression of both WUSCHEL (WUS) and CLAVATA3 (CLV3) genes, which regulate stem cell maintenance in the wild type, were disrupted in l28 shoot apices from early stages on. Unlike in floral patterning, AP2 mRNA is active in the center of the shoot meristem and acts via a mechanism independent of AGAMOUS, which is a repressor of WUS and stem cell maintenance in the floral meristem. Genetic analysis shows that termination of the primary shoot meristem in l28 mutants requires an active CLV signaling pathway, indicating that AP2 functions in stem cell maintenance by modifying the WUS-CLV3 feedback loop.

AGAMOUS Protein, Arabidopsis↗

PubMed2000

Stem cell repopulation efficiency but not pool size is governed by p27(kip1).

Sustained blood cell production requires preservation of a quiescent, multipotential stem cell pool that intermittently gives rise to progenitors with robust proliferative potential. The ability of cells to shift from a highly constrained to a vigorously active proliferative state is critical for maintaining stem cells while providing the responsiveness necessary for host defense. The cyclin-dependent kinase inhibitor (CDKI), p21(cip1/waf1) (p21) dominates stem cell kinetics. Here we report that another CDKI, p27(kip1) (p27), does not affect stem cell number, cell cycling, or self-renewal, but markedly alters progenitor proliferation and pool size. Therefore, distinct CDKIs govern the highly divergent stem and progenitor cell populations. When competitively transplanted, p27-deficient stem cells generate progenitors that eventually dominate blood cell production. Modulating p27 expression in a small number of stem cells may translate into effects on the majority of mature cells, thereby providing a strategy for potentiating the impact of transduced cells in stem cell gene therapy.

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PubMed1997

Impaired steel factor responsiveness differentially affects the detection and long-term maintenance of fetal liver hematopoietic stem cells in vivo.

The results of previous studies have shown that the development of hematopoiesis during fetal life can occur in the absence of Steel factor (SF) signaling. On the other hand, impairment of this mechanism can severely compromise the ability of cells from adult bone marrow to regenerate hematopoiesis on their transplantation into myeloablated recipients. This apparent paradox could result from changes during ontogeny in the responsiveness of hematopoietic stem cells to regulators that may substitute for SF as well as from differences in the availability of such factors during embryogenesis and in the myeloablated adult. To investigate these possibilities, we studied the effect of W41 and W42 mutations on the numbers, phenotype, and posttransplant self-renewal behavior of primitive hematopoietic cells present in the fetal liver (FL) of 14.5-day-old mouse embryos. In W41/ W41 FL, day-12 spleen colony-forming units and long-term culture-initiating cells appeared both quantitatively and qualitatively similar to their counterparts in the FL of +/+ embryos. W41/W41 FL also contained near normal numbers (approximately 50% of controls) of transplantable lymphomyeloid stem cells with competitive reconstituting ability in myeloablated adult +/+ recipients (as assessed for up to at least 16 weeks posttransplant). Moreover, both the original phenotype of these W41/W41 competitive repopulating units (CRUs) and their clonal posttransplant output of mature progeny were normal. Similarly, when myeloablated adult +/+ mice were cotransplanted with 5 x 10(4) +/+ FL cells and a sevenfold to 70-fold excess of W41/W41 FL CRUs, the contribution of the +/+ FL CRUs to the circulating white blood cell count present 5 weeks later was markedly reduced as compared with that of mice that received only +/+ FL cells. However, over the next 3 months, the proportion of mature white blood cells that were derived from +/+ precursors increased significantly (P < .002) in all groups (to > or = 30%), indicating that the ability to sustain hematopoiesis beyond 5 weeks is more SF-dependent than the ability to initially reconstitute both lymphoid and myeloid compartments. Cells from individual FL of W42/+ matings also showed an initial ability (at 7 to 8 weeks posttransplant) to competitively repopulate both lymphoid and myeloid compartments of myeloablated +/+ adult recipients. However, in contrast to recipients of normal or W41/W41 FL cells, the repopulation obtained with the W42 mutant stem cells was transient. Secondary transplants confirmed the inability of the W42 mutant cells to regenerate or even maintain a population of transplantable stem cells. Taken together with previous results from studies of CRUs in adult W mice, these findings support the concept of changes in the way hematopoietic stem cells at different stages of development respond to the stimulatory conditions evoked in the myeloablated recipient. In addition, they provide the first definitive evidence that SF is a limiting physiological regulator of sustained hematopoietic stem cell self-renewal in vivo.

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PubMed1989

Retrovirus-mediated gene transfer to purified hemopoietic stem cells with long-term lympho-myelopoietic repopulating ability.

Despite recent advances in marrow stem cell purification, controversy about the nature and heterogeneity of cells with the potential for long-term repopulation of lymphoid and myeloid tissues remains. Essential to the resolution of these questions is the use of strategies to track the progeny produced in vivo from individual hemopoietic stem cells in purified populations. We have used a procedure for obtaining highly enriched populations of stem cells with competitive repopulating ability from male mice (pretreated with 5-fluorouracil), and in this paper we present the results of studies in which small numbers (150-2000) of these cells were exposed to supernatant containing a helper-free recombinant retrovirus carrying the neomycin-resistance gene and then were transplanted together with 2 x 10(5) "compromised" female marrow cells into irradiated female recipients. Male cells--i.e., progeny of purified stem cells--were found in one or more of the tissues examined (peripheral blood, marrow, spleen, and thymus) in 28 of 28 mice evaluated at various times between 35 and 196 days after transplantation. In 20 of these mice (71%), the neomycin-resistance gene was also detected, although not always at a level that correlated with the proportion of male cells. Analysis of spleen colonies (day 12) generated in secondary recipients confirmed that viral integration was confined to male repopulating cells. In three mice direct evidence of a common clone in both lymphoid and myeloid tissues was also obtained. These results show the feasibility of retrovirus-mediated gene transfer to highly purified populations of lympho-myelopoietic stem cells with long-term (6 months) repopulating potential by using a supernatant infection protocol. This approach should facilitate further analysis of hemopoietic stem cell control in vivo and find future applications in the evolving use of bone marrow transplantation for hemopoietic rescue and gene therapy.

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PubMed1994

Efficient retroviral gene transfer to purified long-term repopulating hematopoietic stem cells.

Efficient gene delivery to multipotential hematopoietic stem cells would greatly facilitate the development of effective gene therapy for certain hematopoietic disorders. We have recently described a rapid multiparameter sorting procedure for significantly enriching stem cells with competitive long-term lymphomyeloid repopulating ability (CRU) from 5-fluorouracil (5-FU)-treated mouse bone marrow. The sorted cells have now been tested as targets for retrovirus-mediated delivery of a marker gene, NeoR. They were cocultured for 4 days with fibroblasts producing a high titer of retrovirus in medium containing combinations of the hematopoietic growth factors interleukin-3 (IL-3), IL-6, c-kit ligand (KL), and leukemia inhibitory factor (LIF) and then injected into lethally irradiated recipients, together with sufficient "compromised" bone marrow cells to provide short-term support. Over 80% of the transplanted mice displayed high levels (> or = 20%) of donor-derived leukocytes when analyzed 4 to 6 months later. Proviral DNA was detected in 87% of these animals and, in half of them, the majority of the hematopoietic cells were marked. Thus, infection of the stem cells was most effective. The tissue and cellular distribution of greater than 100 unique clones in 55 mice showed that most sorted stem cells had lymphoid as well as myeloid repopulating potential. Secondary transplantation provided strong evidence for infection of very primitive stem cells because, in several instances, different secondary recipients displayed in their marrow, spleen, thymus and day 14 spleen colony-forming cells the same proviral integration pattern as the primary recipient. Neither primary engraftment nor marking efficiency varied for stem cells cultured in IL-3 + IL-6, IL-3 + IL-6 + KL, IL-3 + IL-6 + LIF, or all four factors, but those cultured in IL-3 + IL-6 + LIF appeared to have lower secondary engraftment potential. Provirus expression was detected in 72% of the strongly marked mice, albeit often at low levels. Highly efficient retroviral marking of purified lymphomyeloid repopulating stem cells should enhance studies of stem cell biology and facilitate analysis of genes controlling hematopoietic differentiation and transformation.

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PubMed1996

Partially differentiated ex vivo expanded cells accelerate hematologic recovery in myeloablated mice transplanted with highly enriched long-term repopulating stem cells.

The ability of an infusion of ex vivo expanded hematopoietic cells to ameliorate cytopenia following transplantation of hematopoietic stem cells (HSCs) is controversial. To address this issue, we measured the recovery of circulating leukocytes, erythrocytes, and platelets in lethally irradiated mice transplanted with 10(3) enriched HSCs, with or without their expanded equivalent (EE) generated after 7 days of culture in interleukin-3 (IL-3), IL-6, granulocyte colony-stimulating factor and Steel Factor. Two HSC populations differing in their content of short-term repopulating progenitors were evaluated. Thy-1loLIN-Sca-1+ (TLS) bone marrow (BM) is enriched in colony-forming cells (CFCs), day 8 and day 12 spleen colony-forming units (CFU-S) (435 +/- 19, 170 +/- 30, and 740 +/- 70 per 10(3) cells, respectively), and stem cells with competitive long-term repopulating potential (> or = 1 per 43 cells). Thy-1loSca-1+H-2Khl cells (TSHFU) isolated from BM 1 day after treatment of donor mice with 5-fluorouracil (5-FU) are also highly enriched in competitive repopulating units (CRU, > or = 1 per 55 cells), but are depleted of CFCs, day 8 and day 12 CFU-S (171 +/- 8, 0 and 15 +/- 4 per 10(3) cells, respectively). Recipients of 10(3) TLS cells transiently recovered leukocytes to > or = 2,000/microL in 12 days, but sustained engraftment required 25 days. Platelets recovered to > or = 200,000/microL in 15 days, and erythrocytes never decreased below 50% of normal. Mice transplanted with 10(3) TSHFU cells recovered leukocytes in 15 days, and platelets and erythrocytes in 18 days. Recipients of unseparated normal or 5-FU-treated BM cells (containing 10(3) TLS or TSHFU cells) recovered safe levels of blood cells in 9 to 12 days, suggesting that unseparated marrow contains early engrafting cells that were depleted by sorting. Upon ex vivo expansion, total cells, CFCs and day 12 CFU-S were amplified 2,062-,83- and 13-fold, respectively, from TLS cells; and 1,279-, 259- and 708-fold, respectively, from TSHFU cells. Expanded cells could regenerate the majority of lymphocytes and granulocytes in primary (17 weeks) and secondary (26 weeks) hosts and were only moderately impaired compared to fresh HSCs. The EE of TSHFU cells was more potent than that of TLS cells, suggesting that more highly enriched HSCs are more desirable starting populations for this application. When mice were transplanted with 10(3) TSHFU cells and their EE, the duration of thrombocytopenia was shortened from 18 to 12 days, and anemia was abolished. Leukocytes were also elevated on days 9 to 12, although sustained recovery was not accelerated. Anemia was also abrogated in recipients of 10(3) TLS cells and their EE. Early platelet counts were slightly higher than with TLS cells alone, but leukocyte recovery was not improved. These data confirm that TLS cells contribute to early and sustained hematopoiesis, and demonstrate a benefit of ex vivo expanded cells in accelerating engraftment of more primitive TSHFU stem cells depleted of progenitors.

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PubMed1996

The repopulation potential of fetal liver hematopoietic stem cells in mice exceeds that of their liver adult bone marrow counterparts.

Varying, limiting numbers of unseparated or purified cells (Ly-5.1), either from 14.5-day-old fetal liver (FL) or from adult bone marrow (BM) were coinjected with 10(5) unseparated BM cells (Ly-5.2) into lethally irradiated adult C57B1/6 recipients (Ly-5.2). The kinetics of donor cell repopulation of the lymphoid and myeloid compartments by Ly-5.1+ donor hematopoietic stem cells (ie, competitive repopulation units [CRU]) were monitored at various time points after the transplantation by Ly-5 analysis of the peripheral white blood cells (WBC). Recipients that had received on average less than 2 adult BM or FL CRU did not show a significant difference in the level of donor-reconstitution when analyzed 4 weeks after the transplantation, However, at 8 and 16 weeks, the FL recipients showed a significantly higher percentage of donor-derived nucleated peripheral blood cells than did the recipients of adult BM cells. Analysis of individual mice showed that approximately 80% of the recipients of FL CRU showed an increase in mature WBC output between 4 and 8 weeks after transplantation, whereas this occurred in less than 40% in the recipients of adult BM cells. In addition to this effect on mature cell output, the cellularity of the reconstituted BM was significantly higher in recipients of FL CRU than in recipients of adult BM CRU, even at 7 to 9 months after transplantation, which is consistent with an increased clonal expansion of FL CRU. When marrow cells from primary recipients of FL CRU were injected into secondary recipients, a significantly higher percentage of these mice showed donor-reconstitution of their lymphoid and myeloid compartments (P < .01) and to a greater extent (P < .008) as compared with mice that had received marrow cells from primary recipients of similar numbers of adult BM CRU. Taken together, these results show that individual FL CRU exhibit a greater proliferative activity in vivo than similar cells from adult BM that is accompanied by a greater production of daughter CRU.

Animals↗

PubMed2025

Sustained NF-κB activation allows mutant alveolar stem cells to co-opt a regeneration program for tumor initiation.

Disruptions to regulatory signals governing stem cell fate open the pathway to tumorigenesis. To determine how these programs become destabilized, we fate-map thousands of murine wild-type and KrasG12D-mutant alveolar type II (AT2) stem cells in vivo and find evidence for two independent AT2 subpopulations marked by distinct tumorigenic capacities. By combining clonal analyses with single-cell transcriptomics, we unveil striking parallels between lung regeneration and tumorigenesis that implicate Il1r1 as a common activator of AT2 reprogramming. We show that tumor evolution proceeds through the acquisition of lineage infidelity and reversible transitions between mutant states, which, in turn, modulate wild-type AT2 dynamics. Finally, we discover how sustained nuclear factor κB (NF-κB) activation sets tumorigenesis apart from regeneration, allowing mutant cells to subvert differentiation in favor of tumor growth.

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