HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Searles, A. E. Articles by Spangrude, G. J. Search for Related Content PubMed PubMed Citation Articles by Searles, A. E. Articles by Spangrude, G. J. Pubmed/NCBI databases Medline Plus Health Information Bone Marrow Transplantation

Rapid, B Lymphoid-Restricted Engraftment Mediated by a Primitive Bone Marrow Subpopulation¹

A. Elena Searles^*, Suzanne J. Pohlmann, L. Jeanne Pierce^*, S. Scott Perry, William B. Slayton, Mariluz P. Mojica^§ and Gerald J. Spangrude^2,*,

Departments of ^* Oncological Sciences, Pathology, Pediatrics, and ^§ Human Genetics, University of Utah, Salt Lake City, UT 84132

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
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-1.1^low subset as previously described, the Thy-1.1^neg subset consisted of progenitor cells that preferentially reconstituted the B lymphocyte lineage after i.v. transplantation. Recipients of Thy-1.1^neg cells did not survive beyond 30 days, presumably due to the failure of erythroid and platelet lineages to recover after transplants. Thy-1.1^neg 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-1.1^neg population was assayed in culture. When Thy-1.1^low stem cells were rigorously excluded from the Thy-1.1^neg 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-1.1^neg cells was not sustained over a 20-wk period. Therefore, the Thy-1.1^neg population defined in these studies includes transplantable, non-self-renewing B lymphocyte progenitor cells.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
In recent years, investigators studying hematopoietic stem cell (HSC)³ engraftment have shifted their focus toward defining the behavior of these cells shortly after transplant. Although the long-term reconstituting activity of HSCs in murine transplant models has been well documented, there is an increasing interest in better understanding the activity of stem and progenitor cell populations at early times after infusion of a graft. The transition in interest from long- to short-term engraftment in HSC transplantation is partially the result of the need to promote rapid engraftment of functional blood cells in the first few weeks after clinical bone marrow (BM) transplantation. This has led investigators to begin to identify the BM cell populations that contribute to and facilitate early engraftment and to investigate the intrinsic and extrinsic factors that influence the kinetics of early engraftment after BM transplantation (1, 2, 3, 4).

Studies comparing the kinetics of hematopoietic recovery after transplantation of enriched HSCs, as compared with unfractionated BM containing the same number of HSCs, have revealed a marked difference in reconstitutive capabilities of these two populations early after transplantation. In an analysis of the engraftment kinetics of whole BM containing 1000 Sca-1⁺Thy-1.1^lowH-2K^high cells transplanted into irradiated recipients, Szilvassy et al. (5) showed that the unfractionated cells produced a gradual but steady increase in leukocytes through day 30. However, the transplantation of 1000 isolated Sca-1⁺Thy-1.1^lowH-2K^high cells potentiated a sharp and transient increase in leukocyte counts 15 days after transplantation. Leukocyte counts then drastically diminished and eventually reached a normal range around day 30. These studies suggested that regulatory interactions between HSC and other BM cells are lost after cellular enrichment before transplantation, a conclusion that is also supported in studies of allogeneic transplants (6).

Similar studies using a primitive subset of HSC selected based on low retention of the mitochondrial probe rhodamine-123 also demonstrated an early, temporary spike in PBMC counts after transplantation (7). However, analysis of the cells comprising this recovery revealed that they were mainly monocytes and neutrophils and further showed that recovery of the B lymphoid lineage was not observed until about 30 days posttransplant. These results demonstrated that the HSC population within the BM is not capable of rapidly providing early lymphoid reconstitution. Transplantation of purified populations of actively cycling rhodamine-123^high multipotent progenitor cells or of the more lineage-restricted Sca-1^neg progenitor cells revealed that neither possessed the capacity to significantly regenerate leukocytes (7, 8, 9). Thus, lymphocyte progenitor activity observed early after transplantation must be derived from an as yet uncharacterized progenitor cell population.

Interestingly, Okada et al. (10) reported the early recovery (14–21 days) of myeloid and B lymphocytes in the blood after transplantation of highly enriched HSC that were selected based on expression of the c-kit molecule. This observation suggested that a cell population with different functional activities was isolated by this selection protocol compared with previous studies that had isolated HSC based on low levels of Thy-1.1 expression (11). We report here a series of experiments aimed at testing this hypothesis and show that the addition of c-kit expression to the criteria used in previous studies to isolate HSC allows distinction of a committed progenitor population that predominantly generates B lineage cells within 2 wk of i.v. transplantation. By phenotype and function, these progenitors represent a cell population closely related to the common lymphoid progenitor (CLP) recently identified by Kondo et al. (12). However, the lack of selection for the IL-7R in the present studies resulted in significant myeloid lineage potential as well as lymphoid potential in the isolated progenitor population. Interestingly, the use of different fluorochrome conjugates of the c-kit Ab led to variable results, suggesting that fluorochrome conjugates used to isolate particular cell populations can influence the in vivo reconstitutive capabilities of cells to which they bind.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Mice

BM donor animals were 4- to 8-wk-old B6-Thy-1.1-Ly-5.1 or B6.PL (Thy-1.1, Ly-5.2) congenic mice. C57BL/6 (Thy-1.2, Ly-5.2) or B6.SJL (Thy-1.2, Ly-5.1) mice between 8 and 10 wk old served as transplant recipients. Animals were bred and maintained at the Animal Resource Facility at the University of Utah (Salt Lake City, UT) or were purchased from The Jackson Laboratory (Bar Harbor, ME). All animals were maintained on autoclaved, acidified water (pH 2.5) and autoclaved chow.

Harvest and preparation of BM cells for cell sorting

BM was harvested from both femora and tibiae of donor mice. The bones were crushed with a mortar and pestle in HBSS containing 5% FCS and 10 mM HEPES buffer (HBSS/5). After filtering the cells through nylon mesh (pore size, 85 µm; Small Parts, Miami Lakes, FL), cells were washed once with HBSS/5 and centrifuged (1200 rpm at 4°C) for 5 min. The resuspended pellet was treated with ammonium chloride-potassium solution for 2–3 min to lyse RBCs and then was washed again with HBSS/5. To remove mature blood cells from the BM and thereby enrich for rare stem and progenitor cell populations, BM cells were reacted with a lineage (Lin) cocktail containing optimized concentrations of eight mAbs. These mAbs recognize Ags associated with differentiated blood cells and include CD2 (RM-2.2), CD3 (KT3-1.1), CD5 (53-7.3), CD8 (53-6.7), Mac-1 (M1/70), TER-119 (an erythroid Ag), Gr-1 (RB6-8C5), and B220 (RA3-6B2). Cells were incubated with Lin cocktail at a cell density of 5 x 10⁷ cells/ml for 20 min. After the incubation period, cells were washed with HBSS/5 and subsequently incubated with sheep anti-rat Ig-coupled magnetic beads (Dynal, Oslo, Norway). The incubation was performed in a volume of 1 ml at a bead-to-cell ratio of 1:1 with intermittent mixing over a 20-min period. HBSS/5 was then added to bring the volume to 8 ml before magnetic depletion. This depletion process was repeated, and the remaining cells were then incubated with Ly-6A/E (Sca-1, clone D7) mAb conjugated to the fluorochrome PE (PharMingen, San Diego, CA) for 20 min on ice. After a wash, cells were resuspended in HBSS/5 containing 10 µg/ml propidium iodide (PI; Molecular Probes, Eugene, OR) and filtered through nylon mesh.

Flow cytometry

Cells were sorted for Sca-1⁺PI^neg cells using a FACS-Vantage instrument (Becton Dickinson, San Jose, CA) in enrich mode, using a threshold on PE staining as a trigger. Sorted cells were recovered by centrifugation and stained with anti-Thy-1.1 (clone 19XE5, conjugated in our laboratory to Oregon Green, Molecular Probes), PE-Sca-1, and one of two different anti-c-kit reagents. These were either a biotin conjugate (clone ACK4 (13), kindly provided by S.-I. Nishikawa (Kyoto University, Kyoto, Japan) and biotinylated in our laboratory using standard techniques) or an allophycocyanin (APC) conjugate (clone 2B8; PharMingen). The biotin-c-kit reagent was detected with streptavidin-RED613 (Life Technologies, Gaithersburg, MD). After staining, the cells were washed and resuspended in HBSS/5 containing either To-Pro-2 (Molecular Probes) or PI for dead cell exclusion. Cell populations were then sorted using forward scatter triggering in normal recovery mode for Sca-1⁺c-kit⁺ cells, which were further segregated into Thy-1.1^neg and Thy-1.1^low subsets (Fig. 1). Hereafter, the terms "Thy-1.1^neg " and "Thy-1.1^low " are used to indicate Lin^neg populations that are coselected for the Sca-1⁺c-kit⁺ phenotype in addition to the presence or absence of Thy-1.1, as shown in Fig. 1.

View larger version (37K): [in this window] [in a new window]   FIGURE 1. Four-color analyses of mouse BM cells immunomagnetically depleted of mature cells based on expression of CD2, CD3, CD5, CD8, CD19, B220, Gr-1, and TER-119 and gated to include only Sca-1+ cells. These Sca-1+Linneg cells are displayed with respect to c-kit and Thy-1.1 expression. Viability probes used were PI in combination with APC and To-Pro-2 in combination with RED613. Thy-1.1neg and Thy-1.1low cell populations were isolated using gates corresponding to the upper left and upper right quadrants, respectively, of plot A.

Transplantation of sorted cell populations

All recipient mice were exposed to 13 cGy of radiation from a ¹³⁷Cs source (Mark I gamma irradiator; J. L. Shepherd & Associates, Glendale, CA) delivered in two equal doses separated by a 3-h delay. Several hours later, mice were anesthetized using methoxyflurane and were administered purified cells in 0.2 ml HBSS/5 by retroorbital injection. Transplant recipients differed from donors at the Ly-5 locus. For competitive repopulation studies, transplants included 10⁵ syngeneic BM cells in addition to purified congenic cells. Recipient animals were maintained on oral neomycin sulfate (Biosol, 2 mg/ml; Upjohn, Kalamazoo, MI) for 2 wk after irradiation and transplantation.

Analysis of transplant recipients

Engraftment was evaluated by immunofluorescent staining for the Ly-5 allelic Ag on PBMC, BM, and spleen cells at times ranging from 7 to 140 days after the transplants. For PBMC analysis, 150 µl of blood was collected from the retroorbital sinus into a tube containing 20 µl citrate dextrose anticoagulant (Formula A; Baxter Health Care, Mundelein, IL) using heparinized capillary tubes (Fisher Scientific, Pittsburgh, PA). A portion of each blood sample was removed to obtain complete blood counts using an automated hematology analyzer (Serono System 9010; Serono Diagnostics, Allentown, PA). The remainder of the blood was added to a volume of 2% Dextran T500 (Pharmacia Biotech, Piscataway, NJ) in PBS. This mixture was incubated for 30 min in a 37°C water bath to allow for sedimentation of RBCs. After incubation, the upper layer of PBMC was removed from each sample and centrifuged for 5 min at 1200 rpm. The pellet was resuspended in ammonium chloride-potassium solution to lyse residual RBCs and, after a wash in HBSS/5, each sample was aliquoted into three wells of a 96-well microtiter plate. The plate was then centrifuged for 2 min and decanted, and immunofluorescent staining was performed by reacting cells at a density of 5 x 10⁷ cells/ml with saturating solutions of mAb to define and phenotype donor-derived cells based on the allelic difference at the Ly-5 locus. T lymphocyte, B lymphocyte, and myeloid lineages of donor and recipient origin were measured using fluorescein-Ly-5.1 staining in combination with CD4 and CD8 (T lineage), B220 (B lineage), or Mac-1 and Gr-1 (myeloid lineages) mAbs as PE conjugates. Animal groups were set up in duplicate so that no animal was bled more frequently than once per week.

BM and spleen tissues were isolated and single-cell suspensions were made as previously described (7). After RBC lysis, cell counts were determined and cells were reacted with mAb to define and phenotype cells of donor and recipient origin as described above.

Colony-forming assays

CFU content of freshly isolated Thy-1.1^low HSC and Thy-1.1^neg progenitors or of BM tissue after transplantation of these cells was determined by methylcellulose colony assays. Primary CFU activity was evaluated by plating 5 x 10⁴ normal BM cells or 1 x 10² Thy-1.1^low HSC or Thy-1.1^neg progenitors per 35-mm culture plate. Methylcellulose medium consisted of -MEM (Life Technologies), 1.2% methylcellulose (Shinetsu, Tokyo, Japan), 30% FCS (Life Technologies), 1% deionized BSA (Sigma, St. Louis, MO), and 0.1 mM 2-ME (Mallinckrodt Chemical, Chesterfield, MO). Myeloerythroid cultures (CFU-GM and CFU-Mix) were supplemented with the recombinant cytokines IL-3 (10 ng/ml; Peprotech, Rocky Hill, NJ), IL-6 (20 ng/ml; Peprotech), G-CSF (10 ng/ml; Amgen, Thousand Oaks, CA), and erythropoietin (5 U/ml; Ortho Pharmaceuticals, Raritan, NJ). For analysis of secondary CFU activity, lethally irradiated transplant recipients were euthanized 13 days after transplant of 10³ cells, and BM was isolated. Secondary CFU activity was stimulated with IL-3, in the form of 20% WEHI-3B conditioned medium, plus recombinant human erythropoietin (5 U/ml; Ortho Pharmaceuticals). Cells were plated at two densities (5 x 10⁴/ml and 5 x 10⁵/ml) in 35-mm culture plates. All cultures were incubated at 37°C under 5% CO₂, and colonies were enumerated 7–10 days later. CFU-Mix were identified as red colonies by visual inspection or after benzidine staining of hemoglobin. Cultures selective for growth of B lymphocyte progenitors (CFU-preB) contained steel factor (100 ng/ml, a kind gift from Kirin Pharmaceuticals, Tokyo, Japan), Flt3 ligand (75 ng/ml, kindly provided by Immunex, Seattle, WA), and IL-7 (10 ng/ml; Peprotech). Colonies were counted after 8 days.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Definition of two subsets of c-kit⁺ hematopoietic progenitor cells

Previous studies have established that the Sca-1 Ag is expressed by virtually all HSC in the BM of adult B6 mice (8, 9). Furthermore, HSC in adult mouse bone marrow express the Thy-1 Ag in an allele-specific manner, such that in mouse strains expressing Thy-1.1 but not Thy-1.2, HSC are entirely contained within the Thy-1^low subset of BM cells (14). The tyrosine kinase receptor c-kit is a third Ag characteristic of mouse HSC (13). However, the B lymphoid lineage has been reported to engraft within 2 weeks after transplantation of BM progenitors selected on the basis of dual expression of Sca-1 and c-kit (10). In contrast, BM progenitors selected on the basis of Sca-1 and Thy-1.1 expression require 4 wk to regenerate B lymphocytes in the peripheral blood (7). To address this discrepancy, we evaluated the coexpression of Thy-1.1 and c-kit among Sca-1⁺Lin^neg BM cells. As shown in Fig. 1, c-kit⁺Sca-1⁺Lin^neg BM cells were subdivided into two populations of approximately equal frequency by Thy-1.1 staining. Furthermore, cells expressing Thy-1.1 appeared relatively homogeneous for a high level of c-kit expression, whereas those not expressing Thy-1.1 included both c-kit^low and c-kit^high subsets (Fig. 1B). Although equivalent staining patterns and subset frequencies were observed with two different anti-c-kit reagents, an APC-conjugated reagent gave brighter staining and superior resolution of the different levels of c-kit expression compared with a biotin-conjugated reagent that was detected with streptavidin-RED613 (compare Fig. 1, A and B). However, cells selected as c-kit⁺Thy-1.1^neg using the APC conjugate consistently failed to engraft in irradiated recipient animals, whereas the same population selected using the biotin-avidin combination resulted in engraftment after transplantation. Interestingly, the engraftment defect observed when Thy-1.1^neg populations were selected using APC-c-kit was not observed when Thy-1.1^lowc-kit^high HSC were isolated in the same sorts and transplanted into irradiated recipients (data not shown). Consequently, in this paper we report only the transplant experiments performed using the avidin-biotin selection protocol.

Thy-1.1^neg and Thy-1.1^low cell populations differentially reconstitute blood lineages in irradiated animals

After transplantation of 5000 BM c-kit⁺Sca-1⁺Lin^neg cells, selected as either Thy-1.1^neg or Thy-1.1^low, into lethally irradiated recipient animals, we evaluated the recovery of peripheral blood cellularity over time (Fig. 2). In both transplant groups, PBMC counts were below the level of detection (<0.2 x 10⁶/ml) until 12 days after transplantation. Beginning at day 14, recipients of the Thy-1.1^neg subset exhibited highly variable PBMC counts, which in some cases exceeded the values observed in samples obtained from normal animals by severalfold (Fig. 2A). The variability in PBMC counts was to some extent due to the automated hematology counter detecting clumps of activated platelets or circulating normoblasts in the PBMC window, because flow cytometric analysis of the same samples showed few PBMC. In other cases, flow cytometry confirmed high PBMC counts, and phenotypic analysis showed that the majority of circulating PBMC were of the B lymphoid lineage based on a B220⁺ Mac-1/Gr-1^neg phenotype (Fig. 3, A and B). In contrast to recipients of Thy-1.1^neg cells, animals transplanted with Thy-1.1^low cells exhibited a gradual and very consistent increase in PBMC counts beginning at day 12 and reaching near-normal levels by day 30 (Fig. 2A). Phenotypic analysis demonstrated that the majority of cells appearing in the peripheral blood early after transplantation were of the myeloid lineage (Fig. 3, C and D). In one representative experiment, groups of four animals transplanted with either Thy-1.1^neg or Thy-1.1^low cells were evaluated for the frequency of B lymphoid and myeloid cells in peripheral blood 14 days after transplantation. In recipients of Thy-1.1^neg grafts, donor-derived cells in the peripheral blood consisted of (mean ± SD) 87.1 ± 3.5% B lineage cells and 12.8 ± 3.5% myeloid cells. In contrast, recipients of Thy-1.1^low HSC grafts included 16.3 ± 6.8% B lineage cells and 83.3 ± 6.9% myeloid cells among donor-derived peripheral blood cells. In the particular experiment from which the representative phenotypic analysis shown in Fig. 3 was derived, the average PBMC count at 14 days was 2-fold higher in recipients of Thy-1.1^neg cells compared with recipients of Thy-1.1^low cells (Fig. 2A). In absolute numbers, the circulating B lineage cells ranged from 1.3 to 6.7 x 10⁶ cells/ml in recipients of Thy-1.1^neg grafts and from 0.1 to 0.4 x 10⁶ cells/ml in recipients of Thy-1.1^low grafts at day 14.

View larger version (29K): [in this window] [in a new window]   FIGURE 2. Recovery of PBMC (A), RBCs (B), and platelets (C) after transplantation of Thy-1.1neg progenitors (•) or Thy-1.1low HSC () into lethally irradiated recipient mice. The cell populations were enriched as shown in Fig. 1, selecting for c-kit+Sca-1+ cells from lineage-depleted BM and separating these into two subsets based on expression of the Thy-1.1 Ag. The two cell populations were transplanted at a dose of 5000 cells per mouse, and cell counts were performed using a Serono hematology counter. No individual mouse was bled more frequently than once weekly. Data from three separate experiments were pooled and are depicted as mean counts ± SEM. In some cases, error bars were too small to plot.

View larger version (38K): [in this window] [in a new window]   FIGURE 3. Flow cytometric analysis of PBMC recovery 14 days after transplantation of 5000 Thy-1.1low HSC or 5000 Thy-1.1neg progenitor cells. A and B, Analysis of a peripheral blood sample from a recipient of Thy-1.1neg cells. C and D, Analysis of a peripheral blood sample from a recipient of Thy-1.1low cells. In each case, the graft-derived cells are detected by Ly-5 staining and are evaluated as lymphoid lineage by B220 staining (A and C) or as myeloid by Mac-1/Gr-1 staining (B and D). In each case, data representing the entire peripheral blood sample was collected by flow cytometry, so the density of dots is proportional to the PBMC count.

The absence of hematopoietic recovery in the erythroid and platelet lineages in recipients of Thy-1.1^neg grafts is shown in Fig. 2, B and C. Suppression of both lineages was apparent in the first week after radiation conditioning, regardless of whether Thy-1.1^neg or Thy-1.1^low cells were transplanted. The earliest recovery of erythropoiesis after highly enriched HSC grafts has been reported to occur at about day 12 (5, 7), and the results shown in Fig. 2B are consistent with this finding. Irradiated recipients of Thy-1.1^low HSC exhibited prompt erythroid recovery that was maintained throughout the 60-day course of study with a mild degree of anemia. In contrast, animals transplanted with Thy-1.1^neg cells exhibited no erythroid recovery, and by 22 days, RBC counts were 3.33 ± 0.95 x 10⁹/ml (mean ± SD; n = 7) compared with the normal value of 9.79 ± 0.7 x 10⁹/ml (mean ± SD; n = 22). Platelet counts reached a deep nadir in both experimental groups 11 days posttransplant, but by 14 days, recipients of Thy-1.1^low HSC showed evidence of platelet engraftment and platelet counts were within the normal range (1.013 ± 0.339 x 10⁹/ml; mean ± SD, n = 22) by 26 days posttransplant. Recipients of Thy-1.1^neg cells exhibited no evidence of platelet engraftment before death (Fig. 2C).

Differential BM engraftment by subsets of cells defined by Thy-1.1 expression

To test whether preferential engraftment of the B lymphocyte lineage by Thy-1.1^neg cells was due to the reconstitution of the BM with B lineage progenitors, we evaluated recovery of BM 13 days after transplantation of 10³ purified cells. Although equivalent numbers of cells (5.5 x 10⁶ cells/femur) were recovered from transplant recipients of either Thy-1.1^low or Thy-1.1^neg cells, the phenotypic and functional composition of the BM was dramatically different. As shown in Fig. 4, the frequencies of the lymphoid and myeloid components of BM obtained from recipients of Thy-1.1^low HSC closely resembled those observed in BM obtained from normal, untreated mice. In contrast, the balance between lymphoid and myeloid differentiation in the BM samples from recipients of Thy-1.1^neg progenitors was heavily skewed toward the lymphoid lineage, consisting largely of lymphoid cells and including very few cells expressing myeloid Ags (Fig. 4). Consistent with this finding, spleens harvested from the recipient mice revealed 3.0 ± 1.4 splenic colony-forming units (CFU-S) per 100 Thy-1.1^low cells transplanted but fewer than 0.2 CFU-S per 100 Thy-1.1^neg cells.

View larger version (28K): [in this window] [in a new window]   FIGURE 4. Flow cytometric analysis of cellular recovery in the BM after transplantation of 5000 Thy-1.1low HSC or 5000 Thy-1.1neg progenitor cells. BM cells collected 13 days posttransplant were evaluated for expression of the B lineage Ag B220 correlated with the myeloid Ags Mac-1 and Gr-1. Bone marrow cells derived from a normal animal were processed in parallel as a control.

To evaluate the colony-forming potential of Thy-1.1^neg and Thy-1.1^low cells, methylcellulose assays were performed. The results of primary cultures of normal BM cells and each Thy-1.1 subset are shown in Table I. Both subsets included a high frequency of myeloid progenitors compared with unfractionated BM. However, Thy-1.1^low HSC contained 4-fold more CFU-GM and 14-fold more CFU-Mix relative to Thy-1.1^neg progenitor cells. The frequency of CFU-preB was approximately equal in the two subsets and was enriched 700- to 1000-fold compared with normal BM (Table I).

T lineage progenitors are rare among Thy-1.1^neg cells and self-renewal is limited

The failure to observe CFU-preB in BM of mice transplanted with Thy-1.1^neg cells suggests that these cells reconstitute the B lineage progenitor compartment in the BM with non-self-renewing progenitors after transplantation. To further evaluate the potential of Thy-1.1^neg cells to contribute to the lymphoid lineages, a competitive repopulation experiment was performed. Because Thy-1.1^neg cells were incapable of providing protection from lethal doses of radiation, cotransplantation of 10⁵ normal BM cells of Ly-5.2 origin along with 10³ cells of either the Thy-1.1^neg or Thy-1.1^low subsets (Ly-5.1) was performed into lethally irradiated Ly-5.2 recipient mice. The contribution of each Thy-1.1-defined population to peripheral blood lineages was then evaluated over 20 wk, using the Ly-5 allele to distinguish progeny of the BM cells from those of the Thy-1.1 subsets.

The differential contribution of the Thy-1.1^neg and Thy-1.1^low subsets to the platelet lineage was again clearly apparent in these experiments, despite the cotransplanted BM (Fig. 5A). Compared with recipients of Thy-1.1^low cells, recipients of Thy-1.1^neg cells exhibited consistently lower platelet counts but equivalent PBMC counts throughout the 20-wk study period (Fig. 5B). This is consistent with the data shown in Fig. 2, indicating that Thy-1.1^low but not Thy-1.1^neg cells contribute to reconstitution of the platelet lineage. Therefore, the slow platelet recovery observed in recipients of Thy-1.1^neg cells in the competitive transplant assay (Fig. 5A) is due solely to the cotransplanted syngeneic BM, whereas the more rapid platelet recovery seen after transplantation of Thy-1.1^low cells represents the aggregate contributions of both the cotransplanted BM and the Thy-1.1^low HSC. The relative contributions of purified cells vs cotransplanted BM to the platelet lineage were not directly assessed in these experiments because of the lack of Ly-5 expression by platelets.

View larger version (25K): [in this window] [in a new window]   FIGURE 5. Recovery of peripheral blood platelet (A) and PBMC (B) counts after transplantation of 103 Thy-1.1neg or Thy-1.1low HSC in the presence of a competing dose of 105 normal BM cells.

View larger version (21K): [in this window] [in a new window]   FIGURE 6. After transplantation of 103 Thy-1.1neg or Thy-1.1low HSC in the presence of a competing dose of 105 normal BM cells, engraftment of nucleated cells derived from each population was evaluated in the peripheral blood using Ly-5 staining in combination with Ags associated with T lymphoid (A), B lymphoid (B), and myeloid (C) lineages. Absolute numbers of cells were calculated using PBMC counts performed at each timepoint. The limit of detection was defined as 0.5% positive cells at a PBMC count of 106 cells/ml, which corresponds to 5 x 103 donor cells/ml (hatched area in A). Donor-derived T cells were not detected above this level in six of seven recipients of Thy-1.1neg cells. See text for details.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
Although a variety of studies have documented commitment to the B lymphoid lineage and have traced lineage relationships based on expression patterns of cell surface molecules (15, 16), relatively few studies have shown activity of committed B lineage progenitors in transplant experiments (17, 18). Due to the high degree of proliferative potential inherent in uncommitted hematopoietic stem cells, clonal transplant assays can be utilized to demonstrate multilineage engraftment in vivo from single cells (19, 20, 21). In contrast, lineage-specific progenitor cells lack extensive proliferative potential (22). Because of this, lineage-restricted reconstituting activity of hematopoietic progenitor cells has been difficult to demonstrate in transplant models.

A critical consideration in experiments such as those reported in this study is that putative progenitor populations must be isolated in the absence of HSC to demonstrate lineage restriction in nonclonal models. The high proliferative potential of HSC will lead to long-term reconstitution even when a low level of stem cell contamination is present. If early commitment events lead to a restricted representation of lineages in a clone derived from a HSC, the results can be erroneously interpreted as engraftment of a lineage-restricted progenitor. Immunodeficient mouse strains such as severe combined immunodeficiency mutants or recombinase knockouts have been exploited as animal models permissive for lymphoid engraftment in transplant experiments (12, 23, 24). However, the use of immunodeficient mouse strains as transplant recipients may preferentially select for lymphoid engraftment because the nonlymphoid lineages in these animals are normal. This may lead to underestimates of the myeloid and erythroid potentials of transplanted cell populations. In support of this concept, immunodeficient patients who are treated with allogeneic bone marrow transplants often demonstrate mixed chimerism that results in donor-derived lymphoid but not myeloid reconstitution (25, 26). These considerations must be taken into account in transplant studies using early hematopoietic progenitors.

The studies reported in this paper document a transplantable progenitor with restricted lineage potential. The cell population described in these studies has a pattern of surface Ag expression that is very similar to that of HSC, with the exception being a difference in expression of Thy-1.1. Elsewhere we have shown that mouse HSC invariably express Thy-1.1 (14), and it is this lack of HSC contamination among Thy-1.1^neg cells that results in the ability to completely separate the lymphoid progenitor population from multipotent HSC. This separation will not work in mouse strains expressing the Thy-1.2 allele because about 50% of the HSC in these strains lack Thy-1 expression. Contamination of Thy-1.1^neg cell preparations by Thy-1.1^low HSC was noted by sort reanalysis and by functional studies in some of our experiments, underscoring the importance of carefully controlled cell sorting techniques in hematopoietic progenitor separations.

The Thy-1.1^neg progenitor cell population we have characterized largely overlaps with a CLP cell subset previously isolated on the basis of expression of IL-7R (12). In those studies, CLP were selected by a Lin^neg phenotype in combination with low levels of c-kit and Sca-1 expression as well as IL-7R expression. The majority of these cells, which comprised 0.02% of BM, lacked expression of Thy-1.1. The selection described herein utilizes selection for Thy-1.1, Sca-1, and c-kit expression among Lin^neg cells but does not include discrimination based on IL-7R and thus selects a larger population of cells (0.1% of total BM). It is likely that the lack of selection for IL-7R in the present studies resulted in the recovery of myeloid progenitors in addition to lymphoid progenitors because IL-7R expression is limited to the lymphoid lineage. Interestingly, the presence of a significant number of myeloid progenitors among Thy-1.1^neg cells (Table I) did not result in measurable engraftment of platelets, RBCs, or myelomonocytic cells in transplant experiments (Figs. 2, 4, and 6). This may be due to a relative lack of proliferation potential in early myelopoiesis compared with the significant expansion that occurs at the pro-B stage of development. Alternatively, the myeloid progenitors present in the Thy-1.1^neg subset may lack the ability to home to the BM and establish myelopoiesis (Fig. 4). These studies support the concept of an early separation between erythroid and platelet lineages from other myeloid progenitors because these lineages were not observed to recover after transplantation of Thy-1.1^neg cells (Fig. 2) and because erythroid colonies were rarely observed in cultures (Table I). Evidence of a separation of erythroid and platelet progenitors from other myeloid lineages early in hematopoietic development has recently been demonstrated in cell enrichment studies (27).

Kondo et al. (12) utilized clonal analysis to demonstrate that clones generated from single CLP included both T and B lineage potential. However, T lineage potential was evaluated by direct intrathymic injection of individual clones. The low frequency of T cell potential in the Thy-1.1^neg subset isolated in our studies may indicate a defect in thymic homing because our i.v. transplant assay requires both homing, intrathymic proliferation and export of a sufficient number of mature T lymphocytes for detection in the peripheral blood. However, the relevant BM progenitor for T cells is also required to perform these functions to maintain the peripheral pool of T lymphocytes. Therefore, our studies suggest that the cell population that normally seeds the thymus may be a minor subpopulation of the Thy-1.1^neg BM subset, despite the ability of freshly isolated cells or clones derived from those cells to differentiate along the T lineage when injected into the thymus (12). This interpretation is supported by a recent study that shows that B lineage progenitors isolated from Pax5 knockout animals can be reprogrammed to differentiate into multiple hematopoietic lineages, including the T cell lineage (28). It is possible that the in vitro culture utilized by Kondo et al. (12) induced such a lineage reprogramming process in cells that are normally committed to the B lymphoid lineage. Alternatively, segregation of the Thy-1.1^neg population using additional Ags may reveal a small subset with T lineage potential that was transplanted at a frequency too low to be detected in the present studies.

The robust reconstitution of BM and peripheral blood B lineage cells but not other hematopoietic lineages by Thy-1.1^neg cells suggests that this cell population predominantly includes progenitors committed to the B lineage. In separate studies, we have used cell separation techniques and in vitro cultures to show that over 50% of the cells contained in the Thy-1.1^neg subset are committed myeloid progenitors that can be separated from the lymphoid progenitors based on levels of c-kit expression.⁴ Interestingly, myeloid engraftment was minimal in the studies reported in this paper (Figs. 3, 4, and 6), despite the presence of many myeloid progenitors in the transplanted Thy-1.1^neg population (Table I). This suggests that the homing or proliferation of the myeloid progenitor cells contained within the Thy-1.1^neg subset is insufficient to result in significant engraftment. Alternatively, the progeny of the myeloid progenitors may be rapidly sequestered in tissues or may have too short of a lifespan to be detected at the time we evaluated engraftment posttransplant. We assume that the minor degree of myeloid engraftment observed in the present studies (Figs. 3, 4, and 6) was due to the myeloid progenitors contained within the Thy-1.1^neg cell population.

Bauman et al. (29) reported effects of specific Ab detection systems on hematopoietic engraftment analogous to those observed in our studies. In those studies, it was shown that anti-class I MHC Abs detected using an anti-rat secondary reagent inhibited CFU-S activity, whereas anti-class I MHC Abs conjugated to biotin did not have this inhibitory effect. The group concluded that the reduction in CFU-S numbers was due to the phagocytosis of cells opsonized with anti-rat Abs. This did not occur in the case of staining with the avidin-fluorochrome conjugate, possibly because of the presence of fewer Fc regions or because of masking of Fc determinants by the biotin-avidin complexes, which prevented recognition and subsequent phagocytosis of these cells by macrophages. In our studies, a similar mechanism may be causing the preferential removal of cells bound to APC-conjugated Abs. Such a response may be stimulated by the large size and foreign nature of APC (derived from algae), which could activate macrophages to phagocytose Thy-1.1^neg cells opsonized with APC-labeled Abs in a manner similar to that described by Bauman et al. (29). However, the lack of an inhibitory effect of the APC-conjugated c-kit reagent on transplantation of Thy-1.1^low cells, which express very high levels of c-kit (Fig. 1), makes this interpretation difficult to justify. Additional experiments will be required to resolve this question.

The clear clinical value of the Thy1.1^neg cell population in mediating early, albeit transient, PBMC recovery in the posttransplant setting makes it important for us to define the mechanisms that regulate engraftment of this cell population and its ability to facilitate lymphoid reconstitution. In addition, as transplant products are increasingly subjected to in vitro manipulations before infusion to engineer specific balances of graft-vs-leukemia effects with hematopoietic engraftment (30, 31), it will be increasingly important to clarify biologic effects of Ab staining on subsequent transplantation potential.

	Footnotes

 
¹ This work was supported by grants from the National Institutes of Health (RO1 HL56857 and P50 DK49219). The Flow Cytometry and Irradiation core facilities of the Huntsman Cancer Institute, supported by National Cancer Institute Cancer Center Support Grant P30 CA42014, were utilized for these studies.

² Address correspondence and reprint requests to Dr. Gerald Spangrude, 50 North Medical Drive, Room 5C334SOM, Salt Lake City, UT 84132.

³ Abbreviations used in this paper: HSC, hematopoietic stem cell; BM, bone marrow; CLP, common lymphoid progenitor; Lin, lineage; PI, propidium iodide; APC, allophycocyanin; Lin^neg, BM cells depleted of cells expressing any of a panel of lineage-specific Ags; Thy-1.1^neg, Sca-1⁺c-kit⁺Lin^negThy-1.1^neg BM cells; Thy-1.1^low, Sca-1⁺c-kit⁺Lin^negThy-1.1^low BM cells; CFU-GM, colonies containing only granulocytes and macrophages; CFU-Mix, colonies containing both RBC and nucleated cells; CFU-preB, colonies grown under conditions supportive only for B lymphocyte differentiation; CFU-S, colony-forming unit in spleen.

⁴ M. P. Mojica, S. S. Perry, A. E. Searles, K. S. J. Elenitoba-Johnson, L. J. Pierce, A. Wiesmann, W. B. Slayton, and G. J. Spangrude. 2000. Expression of AA4.1 defines the onset of Pax5 transcription in mouse pro-B cells. Submitted for publication.

Received for publication November 12, 1999. Accepted for publication April 12, 2000.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

1. Kaufman, C. L., Y. L. Colson, S. M. Wren, S. Watkins, R. L. Simmons, S. T. Ildstad. 1994. Phenotypic characterization of a novel bone marrow-derived cell that facilitates engraftment of allogeneic bone marrow stem cells. Blood 84:2436.[Abstract/Free Full Text]
2. Papayannopoulou, T., C. Craddock, B. Nakamoto, G. V. Priestley, N. S. Wolf. 1995. The VLA4/VCAM-1 adhesion pathway defines contrasting mechanisms of lodgement of transplanted murine hemopoietic progenitors between bone marrow and spleen. Proc. Natl. Acad. Sci. USA 92:9647.[Abstract/Free Full Text]
3. Peled, A., I. Petit, O. Kollet, M. Magid, T. Ponomaryov, T. Byk, A. Nagler, H. Ben-Hur, A. Many, L. Shultz, et al 1999. Dependence of human stem cell engraftment and repopulation of NOD/SCID mice on CXCR4. Science 283:845.[Abstract/Free Full Text]
4. Shizuru, J. A., L. Jerabek, C. T. Edwards, I. L. Weissman. 1996. Transplantation of purified hematopoietic stem cells: requirements for overcoming the barriers of allogeneic engraftment. Biol. Blood Marrow Transplant. 2:3.[Medline]
5. Szilvassy, S. J., K. P. Weller, B. Chen, C. A. Juttner, A. Tsukamoto, R. Hoffman. 1996. Partially differentiated ex vivo expanded cells accelerate hematologic recovery in myeloablated mice transplanted with highly enriched long-term repopulating stem cells. Blood 88:3642.[Abstract/Free Full Text]
6. Neipp, M., T. Zorina, M. A. Domenick, B. G. Exner, S. T. Ildstad. 1998. Effect of FLT3 ligand and granulocyte colony-stimulating factor on expansion and mobilization of facilitating cells and hematopoietic stem cells in mice: kinetics and repopulating potential. Blood 92:3177.[Abstract/Free Full Text]
7. Nibley, W. E., G. J. Spangrude. 1998. Primitive stem cells alone mediate rapid marrow recovery and multilineage engraftment after transplantation. Bone Marrow Transplant. 21:345.[Medline]
8. Uchida, N., I. L. Weissman. 1992. Searching for hematopoietic stem cells: evidence that Thy-1.1^loLin^-Sca-1⁺ cells are the only stem cells in C57BL/Ka-Thy-1.1 bone marrow. J. Exp. Med. 175:175.[Abstract/Free Full Text]
9. Spangrude, G. J., D. M. Brooks. 1993. Mouse strain variability in the expression of the hematopoietic stem cell antigen Ly-6A/E by bone marrow cells. Blood 82:3327.[Abstract/Free Full Text]
10. Okada, S., K. Nagayoshi, H. Nakauchi, S. I. Nishikawa, Y. Miura, T. Suda. 1993. Sequential analysis of hematopoietic reconstitution achieved by transplantation of hematopoietic stem cells. Blood 81:1720.[Abstract/Free Full Text]
11. Spangrude, G. J., S. Heimfeld, I. L. Weissman. 1988. Purification and characterization of mouse hematopoietic stem cells. Science 241:58.[Abstract/Free Full Text]
12. Kondo, M., I. L. Weissman, K. Akashi. 1997. Identification of clonogenic common lymphoid progenitors in mouse bone marrow. Cell 91:661.[Medline]
13. Ogawa, M., Y. Matsuzaki, S. Nishikawa, S. Hayashi, T. Kunisada, T. Sudo, T. Kina, H. Nakauchi. 1991. Expression and function of c-kit in hemopoietic progenitor cells. J. Exp. Med. 174:63.[Abstract/Free Full Text]
14. Spangrude, G. J., D. M. Brooks. 1992. Phenotypic analysis of mouse hematopoietic stem cells shows a Thy-1-negative subset. Blood 80:1957.[Abstract/Free Full Text]
15. Hayakawa, K., Y. S. Li, R. Wasserman, S. Sauder, S. Shinton, R. R. Hardy. 1997. B lymphocyte developmental lineages. Ann. NY Acad. Sci. 815:15.[Medline]
16. Osmond, D. G., A. Rolink, F. Melchers. 1998. Murine B lymphopoiesis: towards a unified model. Immunol. Today 19:65.[Medline]
17. Kantor, A. B., A. M. Stall, S. Adams, K. Watanabe, L. A. Herzenberg. 1995. De novo development and self-replenishment of B cells. Int. Immunol. 7:55.[Abstract/Free Full Text]
18. Rolink, A., D. Haasner, S. Nishikawa, F. Melchers. 1993. Changes in frequencies of clonable pre B cells during life in different lymphoid organs of mice. Blood 81:2290.[Abstract/Free Full Text]
19. Smith, L. G., I. L. Weissman, S. Heimfeld. 1991. Clonal analysis of hematopoietic stem-cell differentiation in vivo. Proc. Natl. Acad. Sci. USA 88:2788.[Abstract/Free Full Text]
20. Spangrude, G. J., D. M. Brooks, D. B. Tumas. 1995. Long-term repopulation of irradiated mice with limiting numbers of purified hematopoietic stem cells: in vivo expansion of stem cell phenotype but not function. Blood 85:1006.[Abstract/Free Full Text]
21. Morrison, S. J., I. L. Weissman. 1994. The long-term repopulating subset of hematopoietic stem cells is deterministic and isolatable by phenotype. Immunity 1:661.[Medline]
22. McNiece, I. K., I. Bertoncello, A. B. Kriegler, P. J. Quesenberry. 1990. Colony-forming cells with high proliferative potential (HPP-CFC). Int. J. Cell Cloning 8:146.[Medline]
23. Fulop, G. M., R. A. Phillips. 1989. Use of scid mice to identify and quantitate lymphoid-restricted stem cells in long-term bone marrow cultures. Blood 74:1537.[Abstract/Free Full Text]
24. Jr Hackett, J., G. C. Bosma, M. J. Bosma, M. Bennett, V. Kumar. 1986. Transplantable progenitors of natural killer cells are distinct from those of T and B lymphocytes. Proc. Natl. Acad. Sci. USA 83:3427.[Abstract/Free Full Text]
25. van Leeuwen, J. E., M. J. van Tol, A. M. Joosten, P. T. Schellekens, R. L. van den Bergh, J. L. Waaijer, N. J. Oudeman-Gruber, C. P. van der Weijden-Ragas, M. T. Roos, E. J. Gerritsen. 1994. Relationship between patterns of engraftment in peripheral blood and immune reconstitution after allogeneic bone marrow transplantation for (severe) combined immunodeficiency. Blood 84:3936.[Abstract/Free Full Text]
26. Brady, K. A., M. J. Cowan, A. D. Leavitt. 1996. Circulating red cells usually remain of host origin after bone marrow transplantation for severe combined immunodeficiency. Transfusion 36:314.[Medline]
27. Akashi, K., D. Traver, T. Miyamoto, I. L. Weissman. 2000. A clonogenic common myeloid progenitor that gives rise to all myeloid lineages. Nature 404:193.[Medline]
28. Rolink, A. G., S. L. Nutt, F. Melchers, M. Busslinger. 1999. Long-term in vivo reconstitution of T-cell development by Pax5-deficient B-cell progenitors. Nature 401:603.[Medline]
29. Bauman, J. G. J., A. H. Mulder, G. J. van den Engh. 1985. Effect of surface antigen labeling on spleen colony formation: comparison of the indirect immunofluorescence and the biotin-avidin methods. Exp. Hematol. 13:760.[Medline]
30. Bonini, C., G. Ferrari, S. Verzeletti, P. Servida, E. Zappone, L. Ruggieri, M. Ponzoni, S. Rossini, F. Mavilio, C. Traversari, C. Bordignon. 1997. HSV-TK gene transfer into donor lymphocytes for control of allogeneic graft-versus-leukemia. Science 276:1719.[Abstract/Free Full Text]
31. Sehn, L. H., E. P. Alyea, E. Weller, C. Canning, S. Lee, J. Ritz, J. H. Antin, R. J. Soiffer. 1999. Comparative outcomes of T-cell-depleted and non-T-cell-depleted allogeneic bone marrow transplantation for chronic myelogenous leukemia: impact of donor lymphocyte infusion. J. Clin. Oncol. 17:561.[Abstract/Free Full Text]

This article has been cited by other articles:

				S. S. Perry, R. S. Welner, T. Kouro, P. W. Kincade, and X.-H. Sun Primitive lymphoid progenitors in bone marrow with T lineage reconstituting potential. J. Immunol., September 1, 2006; 177(5): 2880 - 2887. [Abstract] [Full Text] [PDF]

				S. E. Prockop and H. T. Petrie Regulation of Thymus Size by Competition for Stromal Niches among Early T Cell Progenitors J. Immunol., August 1, 2004; 173(3): 1604 - 1611. [Abstract] [Full Text] [PDF]

				S. S. Perry, H. Wang, L. J. Pierce, A. M. Yang, S. Tsai, and G. J. Spangrude L-selectin defines a bone marrow analog to the thymic early T-lineage progenitor Blood, April 15, 2004; 103(8): 2990 - 2996. [Abstract] [Full Text] [PDF]

				S. S. Perry, L. J. Pierce, W. B. Slayton, and G. J. Spangrude Characterization of Thymic Progenitors in Adult Mouse Bone Marrow J. Immunol., February 15, 2003; 170(4): 1877 - 1886. [Abstract] [Full Text] [PDF]

				T. Kouro, K. L. Medina, K. Oritani, and P. W. Kincade Characteristics of early murine B-lymphocyte precursors and their direct sensitivity to negative regulators Blood, May 1, 2001; 97(9): 2708 - 2715. [Abstract] [Full Text] [PDF]

				M. P. Mojica, S. S. Perry, A. E. Searles, K. S. J. Elenitoba-Johnson, L. J. Pierce, A. Wiesmann, W. B. Slayton, and G. J. Spangrude Phenotypic Distinction and Functional Characterization of Pro-B Cells in Adult Mouse Bone Marrow J. Immunol., March 1, 2001; 166(5): 3042 - 3051. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Searles, A. E. Articles by Spangrude, G. J. Search for Related Content PubMed PubMed Citation Articles by Searles, A. E. Articles by Spangrude, G. J. Pubmed/NCBI databases Medline Plus Health Information Bone Marrow Transplantation