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Copper chelation has been shown to favor the expansion of human hematopoietic stem/progenitor cells in vitro. To further understand the effects of copper modulation on defined subsets of stem cells versus progenitor cells, we extended the studies in a mouse system. We isolated mouse hematopoietic stem cells (HSCs) or hematopoietic progenitor cells (HPCs) and cultured them with or without the copper chelator tetraethylenepentamine (TEPA) or CuCl2. Cytokine-stimulated HPC cultures treated with TEPA for 7 days generated about two to three times more total and erythroid colony-forming cells (CFCs) compared to control cultures. In contrast, CuCl2treatment decreased the CFC numbers. Similar results were seen with HSC after 14, but not 7, days of culture. Transplant studies showed that HPCs cultured for 7 days in TEPA had about twofold higher short-term erythroid repopulation potential compared to control cultures, while CuCl2decreased the erythroid potential of cultured HPCs compared to control cultures. HSCs cultured with TEPA for 7 days did not exhibit significantly higher repopulation potential in either leukocyte or erythrocyte lineages compared to control cultures in short-term or long-term assays. Based on JC-1 staining, the mitochondrial membrane potential of HPCs cultured with TEPA was lower relative to control cultures. Our data suggest that decreasing the cellular copper content with TEPA results in preferential expansion or maintenance of HPC that are biased for erythroid differentiation in vivo, but does not enhance the maintenance of HSC activity in culture.

Objective The OP9-DL1 culture system is an in vitro model for T-cell development in which activation of the Notch pathway by Delta-like 1 promotes differentiation of mature T cells from progenitors. The roles of specific cytokines in this culture system have not been well defined, and controversy regarding the role of IL-7 has recently emerged. We examined the roles played by IL-7, Flt3 ligand, and stem cell factor (SCF) in differentiation of adult bone marrow cells in the OP9-DL1 culture system. Methods Hematopoietic progenitor cells isolated from mouse bone marrow were cultured with OP9 or OP9-DL1 stromal cells and evaluated for T and B lymphocyte differentiation using immunofluorescent staining. Results IL-7 provided both survival/proliferation and differentiation signals in a dose-dependent manner. T-cell development from the CD4/CD8 double-negative (DN) stage to the CD4/CD8 double-positive (DP) stage required IL-7 provided by the stromal cells, while differentiation from the DP to the CD8 single-positive (SP) stage required addition of exogenous IL-7. SCF favored the proliferation of DN lymphoid progenitors and inhibited differentiation to the DP stage in a dose-dependent manner. Conversely, blocking the function of SCF expressed endogenously by OP9-DL1 cells inhibited proliferation of lymphoid progenitors and accelerated T-lineage differentiation. Flt3 ligand promoted proliferation without affecting differentiation. Conclusion These results validate the OP9-DL1 model for the analysis of T-cell development from bone marrow–derived progenitor cells, and demonstrate specific roles of SCF, IL-7, and Flt3L in promoting efficient T-lineage differentiation.

We have recently described a subset of the multipotent progenitor pool that contains a common lymphoid progenitor. This subset of cells is lineage negative and expresses c-kit and Sca-1, but lacks expression of Thy 1.1 (Thyneg). Based on the observation that lethally irradiated mice transplanted with these cells die from anemia unless supported with competitor marrow, we hypothesized that these progenitors lacked erythroid potential. We analyzed the erythroid potential of these cells by transplanting them into mice allelic at the hemoglobin locus and compared their erythroid potential with the Thy-1.1low (Thylow) subset that contains hematopoietic stem cells. We also performed CFU-C assays in methylcellulose containing recombinant cytokines and determined erythroid contribution to colonies using in situ benzidine staining. Donor-derived hemoglobin was observed following transplant of Thyneg cells, even though 19 of 20 of these animals died from anemia. In contrast, recipients of Thylow cells showed complete donor-derived engraftment 30 days following transplant. While approximately 60% of day 4 colonies derived from Thyneg cells expressed hemoglobin, by day 11 less than 5% were hemoglobinized. In contrast, greater than 70% of the Thylow subset contained hemoglobinized cells at the end of the observation period. A similar transient appearance of myeloid progeny was also observed in colonies derived from c-kitlow Thyneg lymphoid progenitor cells. We conclude that these studies demonstrate commitment to the lymphoid lineage at the Thylow-to-Thyneg interface, and that the loss of erythroid and myeloid potential is gradual rather than abrupt. Hemoglobinized colonies may be undergoing apoptosis because of down-regulation of GATA-1 or because of a death signal from surrounding nonerythrocytic cells.

The stem cell pool can be fractionated by using the mitochondrial dye, rhodamine-123, into Rho(low) hematopoietic stem cells and Rho(high) progenitors. Rho(low) stem cells permanently engraft all lineages, whereas Rho(high) progenitors transiently produce erythrocytes, without substantial platelet or granulocyte production. We hypothesized that the inability of the Rho(high) cells to produce platelets in vivo was due to the fact that these cells preferentially engraft in the spleen and lack marrow engraftment. Initially, we demonstrated that Rho(high) progenitors produced more megakaryocytes in vitro than Rho(low) stem cells did. To study the activity of the Rho(low) and Rho(high) subsets in vivo, we used mice allelic at the hemoglobin and glucose phosphate isomerase loci to track donor-derived erythropoiesis and thrombopoiesis. Rho(low) stem cells contributed to robust and long-term erythroid and platelet engraftment, whereas Rho(high) progenitors contributed only to transient erythroid engraftment and produced very low numbers of platelets in vivo. Donor-derived megakaryopoiesis occurred at higher densities in the spleen than in the bone marrow in animals receiving Rho(low) stem cells and peaked around day 28. Blockade of splenic engraftment using pertussis toxin did not affect the peak of splenic megakaryopoiesis, supporting the hypothesis that these megakaryocytes were derived from progenitors that originated in the bone marrow. These data emphasize that in vitro behavior of hematopoietic progenitor cell subsets does not always predict their behavior following transplantation. This study supports a major role for the spleen in thrombopoiesis following engraftment of transplanted stem cells in irradiated mice.

Utilizing multiparameter flow cytometry, we have defined a subset of bone marrow cells containing lymphoid-restricted differentiation potential after i.v. transplantation. Bone marrow cells characterized by expression of the Sca-1 and c-kit Ags and lacking Ags of differentiating lineages were segregated into subsets based on allele-specific Thy-1.1 Ag expression. Although hematopoietic stem cells were recovered in the Thy-1.1low subset as previously described, the Thy-1.1neg subset consisted of progenitor cells that preferentially reconstituted the B lymphocyte lineage after i.v. transplantation. Recipients of Thy-1.1neg cells did not survive beyond 30 days, presumably due to the failure of erythroid and platelet lineages to recover after transplants. Thy-1.1neg cells predominantly reconstituted the bone marrow and peripheral blood of lethally irradiated recipients with B lineage cells within 2 weeks, although a low frequency of myeloid lineage cells was also detected. In contrast, myeloid progenitors outnumbered lymphoid progenitors when the Thy-1.1neg population was assayed in culture. When Thy-1.1low stem cells were rigorously excluded from the Thy-1.1neg subset, reconstitution of T lymphocytes was rarely observed in peripheral blood after i.v. transplantation. Competitive repopulation studies showed that the B lymphoid reconstitution derived from Thy-1.1neg cells was not sustained over a 20-wk period. Therefore, the Thy-1.1neg population defined in these studies includes transplantable, non-self-renewing B lymphocyte progenitor cells.

Hematopoietic stem cells (HSC) are defined by self-renewal and multilineage differentiation potentials. In order to uncover the genetic program of HSC, we utilized high-density arrays to compare gene expression in highly purified mouse HSC and their mature progeny. One molecule specifically expressed in immature cells is CD27, a member of the TNF receptor family previously shown to play roles in lymphoid proliferation, differentiation, and apoptosis. We show here that the CD27 protein is expressed by about 90% of cells in a purified HSC population. Interestingly, the CD27pos cells are enriched for cells with short-term hematopoietic activities (colony forming potential in vivo and in vitro), while the minority CD27neg population is more effective in clonal long-term transplantation.

Transgenic expression of a gain-of-function truncated mouse erythropoietin receptor gene (EpoR) leads to expansion of the HSC pool in response to human erythropoietin (Epo). We have re-examined this observation using a knock-in mouse model, wherein the mouse EpoR gene was replaced in its proper genetic locus by a single copy of either a wild-type human or a polycythemia-inducing truncated human EPOR gene. Bone marrow cells obtained from knock-in mice were transplanted together with competitor bone marrow cells in a model that allows tracking of erythroid, platelet, and leukocyte contributions by each genotype. Secondary transplants were also performed. Stem/progenitor cells were identified phenotypically and isolated for colony-forming assays to evaluate cytokine responsiveness by cells with the wild-type human or truncated human EPOR gene. Augmented Epo signaling increased erythroid repopulation post-transplant as expected, but had no effect on short-term or long-term leukocyte repopulation. However, the wild-type human EPOR knock-in mouse showed decreases in both erythroid and platelet repopulation compared to marrow cells from the mutant human EPOR knock-in mouse or normal B6 animals. These results provide evidence supporting a role for Epo signaling in megakaryopoiesis in vivo and suggest a role for Epo signaling early in hematopoietic development.

Bone marrow contains a heterogeneous mixture of mature and maturing precursors of blood cells, progenitor cells for myeloid and lymphoid lineages, and hematopoietic and mesenchymal stem cells. The differentiation potential of these different stem, progenitor, and precursor populations can be evaluated by using transplantation and cell culture assays. In this study, we used a stromal cell co-culture system to evaluate the B and T lineage potential of different subsets of mouse bone marrow. We enriched hematopoietic stem (Lin - Sca-1 + c-kit + Thy1.1 l o w [Thy1.1 l o w ]) cells and lymphoid progenitor (Lin - Sca-1 + c-kit + Thy1.1 - [Thy1.1 - ]) cells from mouse bone marrow and co-cultured these populations with OP9 or OP9-DL1 stromal cell lines. Development of the B and T lineages was evaluated over time. Both populations gave rise to B and T cells but with different kinetics. Thy1.1 - lymphoid progenitors gave rise to B and T lineage cells earlier than did Thy1.1 l o w stem cells; and at any given time, percentages of differentiating B and T cells were higher in Thy1.1 - cultures than in Thy1.1 l o w cultures. We also compared the lineage potential of Thy-1.1 - lymphoid progenitors with that of the recently described common lymphoid progenitor 2 (isolated as Lin - Sca-1 + c-kit-Thy1.1 - B220 + cells [B220 + ]). B220 + cells produced B lineage progeny in OP9 cultures more rapidly than did Thy1.1 - cells and produced higher percentages of differentiating T cells in OP9-DL1 cultures. These studies demonstrate the utility of the OP9 and OP9-DL1 co-culture systems for evaluation of lymphoid lineage potential and for determining the relative position of specific bone marrow populations within the hematopoietic hierarchy.

The recent description of an early T-lineage progenitor (ETP) population in adult mouse thymus implies the presence of a bone marrow predecessor that has not yet been identified. Here we describe a LinNeg Sca-1Pos c-kitHi Thy-1.1Neg L-selectinPos adult mouse bone marrow population that resembles the thymic ETP in both antigen expression phenotype and posttransplantation lineage potential. These cells produce wavelike kinetics of thymic seeding and reconstitute the irradiated thymus with kinetics comparable to a thymocyte graft after intravenous transplantation. Transient B-lineage reconstitution is also observed, but little myeloid potential can be detected in transplant experiments. A second subset of progenitors is L-selectinNeg and is highly enriched for rapid and persistent T- and B-lineage potential, as well as some myeloid potential. L-selectin (CD62L) is therefore an effective marker for separating lymphoid progenitors from myeloid progenitors and hematopoietic stem cells in mouse bone marrow. (Blood. 2004;103: 2990-2996)

Thymic cellularity is maintained throughout life by progenitor cells originating in the bone marrow. In this study, we describe adult mouse bone cells that exhibit several features characteristic of prothymocytes. These include 1) rapid thymic engraftment kinetics following i.v. transplantation, 2) dramatic expansion of thymic progeny, and 3) limited production of hemopoietic progeny other than thymocytes. The adult mouse bone marrow population that is depleted of cells expressing any of a panel of lineage-specific Ags, stem cell Ag-1 positive, and not expressing the Thy1.1 Ag (Thy1.1−) (Thy1.1− progenitors) can repopulate the thymus 9 days more rapidly than can hemopoietic stem cells, a rate of thymic repopulation approaching that observed with transplanted thymocytes. Additionally, Thy1.1− progenitors expand prolifically to generate thymocyte progeny comparable in absolute numbers to those observed from parallel hemopoietic stem cell transplants, and provide a source of progenitors that spans multiple waves of thymic seeding. Nevertheless, the Thy1.1− population yields relatively few B cells and rare myeloid progeny posttransplant. These observations describe the phenotype of an adult mouse bone marrow population highly enriched for rapidly engrafting, long-term thymocyte progenitors. Furthermore, they note disparity in B and T cell expansion from this lymphoid progenitor population and suggest that it contains the progenitor primarily responsible for seeding the thymus throughout life.

The induction of apoptosis during cytokine-induced proliferation of hematopoietic stem and progenitor cells (HSPC) may result in the loss of hematopoietic function. We tested the ability of several caspase inhibitors to maintain transplantation potential of mouse HSPC during in vitro culture. HSPC were isolated from mouse bone marrow by cell sorting and cultured in the presence of steel factor (STL) with or without various caspase inhibitors. After incubation, cells were harvested and tested for in vitro colony-forming cell (CFC) potential and transplantation activity in both short- and long-term in vivo assays. HSPC required STL to retain CFC activity during a 24-h culture at 37 degrees C, and none of three caspase inhibitors could substitute for STL in this respect. In transplant assays, a twofold higher frequency of animals showed donor-derived blood cells 12 weeks after competitive transplantation of 50 HSPC cultured for 4 h in the presence of STL plus n-acetyl-Tyr-Val-Ala-Asp-chloromethyl ketone (ac-YVAD) compared with 50 cells cultured in STL alone. To evaluate the effect of ac-YVAD on short-term engraftment, 500 cultured HSPC were transplanted into lethally irradiated mice. Animals transplanted with cells cultured in the presence of ac-YVAD showed a higher survival rate and a faster recovery of platelets and hematocrit compared with animals transplanted with cells cultured in STL alone. We conclude that both the short-term and the long-term engraftment potentials of HSPC cultured in the presence of STL + ac-YVAD were superior to that obtained from cells cultured in STL alone.

The induction of apoptosis during cytokine-induced proliferation of hematopoietic stem and progenitor cells (HSPC) may result in the loss of hematopoietic function. We tested the ability of several caspase inhibitors to maintain transplantation potential of mouse HSPC during in vitro culture. HSPC were isolated from mouse bone marrow by cell sorting and cultured in the presence of steel factor (STL) with or without various caspase inhibitors. After incubation, cells were harvested and tested for in vitro colony-forming cell (CFC) potential and transplantation activity in both short- and long-term in vivo assays. HSPC required STL to retain CFC activity during a 24-h culture at 37°C, and none of three caspase inhibitors could substitute for STL in this respect. In transplant assays, a twofold higher frequency of animals showed donor-derived blood cells 12 weeks after competitive transplantation of 50 HSPC cultured for 4 h in the presence of STL plus n-acetyl-Tyr-Val-Ala-Asp-chloromethyl ketone (ac-YVAD) compared with 50 cells cultured in STL alone. To evaluate the effect of ac-YVAD on short-term engraftment, 500 cultured HSPC were transplanted into lethally irradiated mice. Animals transplanted with cells cultured in the presence of ac-YVAD showed a higher survival rate and a faster recovery of platelets and hematocrit compared with animals transplanted with cells cultured in STL alone. We conclude that both the short-term and the long-term engraftment potentials of HSPC cultured in the presence of STL + ac-YVAD were superior to that obtained from cells cultured in STL alone.

A lymphoid-committed progenitor population was isolated from mouse bone marrow based on the cell surface phenotype Thy-1.1negSca-1posc-KitlowLinneg. These cells were CD43posCD24pos on isolation and proliferated in response to the cytokine combination of steel factor, IL-7, and Flt3 ligand. Lymphoid-committed progenitors could be segregated into more primitive and more differentiated subsets based on expression of AA4.1. The more differentiated subset generated only B lymphoid cells in 92% of total colonies assayed, lacked T lineage potential, and expressed Pax5. These studies have therefore defined and isolated a B lymphoid-committed progenitor population at a developmental stage corresponding to the initial expression of CD45R.

The ability to study lymphocyte differentiation in culture has been greatly advanced by the availability of the OP9 bone marrow stromal cell line, which was derived from an op/op mouse and thus lacks M-CSF. As a result, the normal default myeloid differentiation from bone marrow-derived stem and progenitor cells does not occur, and lymphocyte differentiation is favored. Introduction of the Notch ligand Delta-like 1 into OP9 cells results in promotion of T cell development and parallel suppression of B cell development. While the OP9-DL1 model of T cell development works quite well when fetal liver-derived progenitors are cultured, the success of T cell development from adult bone marrow-derived progenitors has been more difficult to reproduce. We have undertaken a systematic analysis of variables that can prevent efficient T cell development in OP9-DL1 cultures, and have found that one limiting factor that impacts the efficiency of differentiation of both T and B cell lineages is the accumulation of ammonium ions as a result of the spontaneous decomposition of l-glutamine. L-glutamine, which is present at 2 to 4 mM in standard tissue culture media, is unstable and will spontaneously degrade to form ammonium ions and pyroglutamic acid at a rate of 1%/day at 4°C and at a 10-fold higher rate at 37°C. To evaluate the effects of the two major products of l-glutamine decomposition on lymphoid differentiation, we added each product to differentiation cultures at 3 mM in the presence of a stable source of l-glutamine (l-alanyl-l-glutamine). Cultures were established in 1 ml containing 4×104 stromal cells (OP9 for B cell differentiation, OP9-DL1 for T cell differentiation), 1×103 bone marrow-derived lymphoid progenitors enriched by phenotype (c-kit+LinnegSca-1+Thy-1.1neg), and 5 ng/ml Flt3L plus 5 ng/ml IL-7. Every 3 to 4 days, cultures were harvested and passaged onto fresh stromal cell monolayers; lymphoid cells were counted and evaluated for surface antigen expression at each passage. While addition of pyroglutamic acid had no inhibitory effect on lymphocyte growth or differentiation, addition of ammonium chloride slowed growth and prevented differentiation of both T and B lymphocytes. Growth of the stromal cell monolayers was not affected by ammonium chloride at the concentrations utilized in these studies. We conclude that freshly-prepared culture medium, preferably containing a stabilized form of l-glutamine, is a critical aspect contributing to the success of lymphocyte differentiation cultures established from adult bone marrow cells. We also found that decreasing IL-7 concentrations to 1 ng/ml resulted in more rapid differentiation of T cells and a more balanced representation of CD4 and CD8 single positive cells. Our studies help define optimal conditions for differentiation of bone marrow-derived lymphoid progenitor cells into T and B lineages in vitro, and provide evidence that hematopoietic differentiation displays variable degrees of sensitivity to ammonium ions derived from decomposition of l-glutamine. These results will help define optimal conditions for expansion and differentiation of hematopoietic stem and progenitor cells in vitro.

We have developed a method to clonally mark hematopoietic stem and lymphoid progenitor cell populations using a novel sequence tag approach. A library containing an 11-base random sequence tag is cloned into a lentivirus vector, packaged using the VSV-G glycoprotein and HIV-1 capsid, and transduced into freshly isolated mouse hematopoietic stem cell (Thy-1.1lowc-kithigh) or progenitor cell (Thy-1.1negc-kithigh) populations. To minimize artifacts introduced by prolonged culture, we have utilized a 3-hour spinoculation protocol performed in the absence of cytokines. Transduction efficiency was evaluated in vitro by methylcellulose colony assay and liquid cultures, and in vivo by transplanting the transduced cells into lethally irradiated mice. A bicistronic lentivirus vector with a CMV promoter driving expression of a transcript encoding Thy-1.2-IRES-GFP was used to optimize the transduction protocol. Liquid culture assays demonstrated 57% transduction efficiency after 5 days of growth, based on expression of the Thy-1.2 and GFP reporter proteins. Mice transplanted with transduced Thy-1.1negc-kithigh progenitor cells were sacrificed after 16 days, a time at which we have previously observed robust progenitor cell engraftment in the thymus while progeny of Thy-1.1lowc-kithigh HSC have not yet appeared. In 4 of 4 transplanted mice, we observed donor-derived cells in the bone marrow, lymph nodes and thymus. The percentage of total cells expressing the lentivirus-derived transgene ranged from 1.6% of bone marrow cells to 20% of thymocytes. Peripheral blood from mice transplanted with transduced HSC were analyzed and monitored every 4 weeks for transgene expression. We observed that although the Thy-1.2 marker was expressed and maintained up to 14 weeks after HSC transplant, GFP transgene expression was minimal. Based on these preliminary results, we have engineered a new lentivirus vector containing random sequence tags and the Thy-1.2 marker. This strategy provides a simple and efficient way of tracking the progeny of individual cells within a transplanted population, using PCR amplification of the random tags found within mature cell populations derived from the transduced cells. Sequence analysis of individual clones derived from different lineages of cells will enable us to better define the lineage potentials of specific progenitor cell subpopulations.

Notch signaling plays a critical role in T lineage commitment during lymphoid differentiation. However, Notch signaling alone is not sufficient to support T cell development through the CD4/CD8 double positive (DP) stage in vitro. We here report distinct effects of several cytokines on T cell differentiation in the OP9-DL1 cell culture model. Our studies show that Flt3 ligand enhances the proliferation of progenitors but has no obvious effect on differentiation. In contrast, stem cell factor (SCF) favors the proliferation of CD4/CD8 double negative (DN) lymphoid progenitors and inhibits differentiation to the DP stage in a dose-dependent manner. Differentiation of the NK lineage is promoted under these conditions. Conversely, blocking the function of SCF that is expressed endogenously by OP9-DL1 cells inhibits proliferation of lymphoid progenitors and accelerates T lineage differentiation. IL-7 is necessary for differentiation from the DP to the CD8 single positive (SP) stage, and is also required for γδ T lineage development. We also find a dosage effect of IL-7 during T cell development. OP9 and OP9-DL1 stromal cells produce endogenous levels of IL-7 that are sufficient to support B and DP T cell differentiation. However, the amount of endogenous IL-7 is not sufficient to support T cell differentiation from the DP to the SP stage. Addition of exogenous IL-7 (1–10 ng/ml) to the cultures promotes SP differentiation, while blocking endogenous IL-7 with anti-IL-7 antibody inhibits both B and T cell development. We conclude that activation through the Notch pathway is sufficient to suppress B lineage differentiation and thereby promote T lineage commitment, but is not sufficient to promote the subsequent stages of T cell development. SCF promotes expansion and directs NK lineage differentiation at the expense of T cell development, while IL-7 provides both proliferation as well as T lineage differentiation signals. T cell development from the DN to the DP stage requires a low amount of IL-7, while differentiation from the DP to the SP stage requires a higher level of IL-7. The balance between the effects mediated by these cytokines, along with Notch signaling, plays a critical role in regulating development of the T and NK lineages.

We have recently described a mouse bone marrow population defined by the phenotype Thy1.1 Neg , Sca1 Pos , Lin Neg (Thy Neg ) which contains lymphoid progenitor cells. To evaluate the engraftment kinetics of these progenitors in comparison to Thy1.1 Low , Sca1 Pos , Lin Neg hematopoietic stem/progenitor cells (HSPC), each population was transplanted intravenously into sublethally irradiated (6.5 Gy) Ly5 congenic mice at 10 3 cells per mouse. Over a course of 14 to 28 days post-transplant, both thymic lobes and one femur from each mouse were collected and assayed for the presence of donor cells by immunofluorescent staining and flow cytometry. Engraftment of CD19 Pos B cells was observed in bone marrow of Thy Neg recipients by day 14, three days earlier than recipients of HSPC. Myeloid engraftment (Gr-1 Pos ) was also observed in HSPC transplant recipients by day 17, but was not observed in Thy Neg recipients. Thymic reconstitution was observed at significant levels (>10 6 cells per lobe) in 25% of Thy Neg recipients by day 14. In contrast, donor-derived thymocytes were not observed in recipients of HSPC until day 21, when 1 of 20 thymic lobes was positive for significant engraftment compared to 9 of 24 lobes assayed from Thy Neg recipients. HSPC engraftment proceeded rapidly after day 21, and by day 28 all of the lobes assayed were positive for >10 6 donor-derived thymocytes. Thy Neg recipients achieved this degree of engraftment in only 33% of lobes assayed. We conclude from these observations that the Thy Neg population contains lymphoid progenitors capable of more rapid thymic engraftment relative to HSPCs. This suggests that specific commitment steps have predisposed these cells to thymic homing and engraftment. The difference in engraftment kinetics will allow us to distinguish the activity of lymphoid progenitors from HSPCs. This assay will facilitate enrichment and characterization of bone marrow-derived T cell progenitors.