The Journal of Experimental Medicine
JEM © The Rockefeller University Press $8.00
Vol. 203, No. 1, January 23, 2006 73–85 www.jem.org/cgi/doi/10.1084/jem.20051714
As a population, hematopoietic stem cells
(HSCs) have the remarkable ability to drive
hematopoiesis for the lifetime of the organism
while maintaining and even expanding their
numbers with age (1). The capacity of stem
cells to both self-renew and yet maintain
multilineage potential appears to be dependent
on the localization of HSCs within specifi c
microenvironments within the bone marrow
because other organs associated with extra-
medullary hematopoiesis, such as the spleen,
cannot maintain the self-renewal potential of
HSCs (2). Although the molecular and cellular
details of these HSC niches are only just
beginning to be revealed (3–6), in normal
settings it is likely that these specialized micro-
environments are as rare as the HSCs they sup-
port. Another hallmark property of HSCs is
their intrinsic ability to home to these very
specifi c niches within the bone marrow after
intravenous transplantation. Upon adoptive trans -
fer into myeloablated recipients, single HSCs
can give rise to all of the blood lineages for the
lifetime of the recipient (7, 8). Transplanta -
tion into unconditioned recipients, how ever,
has led to mixed results. Transplantation of ex-
traordinarily high doses of unfractionated con-
genic bone marrow or populations enriched
for HSCs has led to hematopoietic chimerism
(9, 10), although it is unclear whether this do-
nor contribution is indicative of HSC engraft-
ment and multilineage reconstitution, or solely
the survival of lymphoid cells, which can persist
for long periods of time in the absence of fur-
ther input from the bone marrow (11–13). In
contrast, conventional intravenous transplan-
tation of more moderate amounts of conge-
nically marked syngeneic or allogeneic bone
mar row or purifi ed HSCs rarely, if ever, leads
to sustained multilineage engraftment without
irradiation of the recipient (14, 15). Thus, the
bulk of evidence has suggested that HSC niches
are fi lled under normal conditions.
Recent evidence, however, has suggested
that a small fraction of HSC niches may be
available in normal animals. First, ?100 HSCs
<doi>10.1084/jem.20051714</doi><aid>20051714</aid>Purifi ed hematopoietic stem cell engraftment
of rare niches corrects severe lymphoid
defi ciencies without host conditioning
Deepta Bhattacharya, Derrick J. Rossi, David Bryder,
and Irving L. Weissman
Department of Pathology, Institute of Cancer and Stem Cell Biology and Medicine, Stanford University School of Medicine,
Stanford, CA 94305
In the absence of irradiation or other cytoreductive conditioning, endogenous hematopoi-
etic stem cells (HSCs) are thought to fi ll the unique niches within the bone marrow that
allow maintenance of full hematopoietic potential and thus prevent productive engraft-
ment of transplanted donor HSCs. By transplantation of purifi ed exogenous HSCs into
unconditioned congenic histocompatible strains of mice, we show that ? ?0.1–1.0% of these
HSC niches are available for engraftment at any given point and fi nd no evidence that
endogenous HSCs can be displaced from the niches they occupy. We demonstrate that
productive engraftment of HSCs within these empty niches is inhibited by host CD4+ T cells
that recognize very subtle minor histocompatibility differences. Strikingly, transplantation
of purifi ed HSCs into a panel of severe combined immunodefi cient (SCID) mice leads to a
rapid and complete rescue of lymphoid defi ciencies through engraftment of these very rare
niches and expansion of donor lymphoid progenitors. We further demonstrate that tran-
sient antibody-mediated depletion of CD4+ T cells allows short-term HSC engraftment and
regeneration of B cells in a mouse model of B(-) non-SCID. These experiments provide a
general mechanism by which transplanted HSCs can correct hematopoietic defi ciencies
without any host conditioning or with only highly specifi c and transient lymphoablation.
deepta @ stanford.edu deepta @ stanford.edu
Abbreviations used: APC, allo-
phycocyanin; CLP, common
lymphoid progenitor; CMP,
common myeloid progenitor;
GMP, granulocyte macrophage
progenitor; GVHD, graft-versus-
host disease; HSC, hematopoi-
etic stem cell; LT, long-term;
MEP, megakaryocyte erythro-
cyte progenitor; NP,
Abbreviations used: APC, allo-
phycocyanin; CLP, common
lymphoid progenitor; CMP,
common myeloid progenitor;
GMP, granulocyte macrophage
progenitor; GVHD, graft-versus-
host disease; HSC, hematopoi-
etic stem cell; LT, long-term;
MEP, megakaryocyte erythro-
cyte progenitor; NP,
74 ENGRAFTMENT IN RARE EMPTY HEMATOPOIETIC STEM CELL NICHES | Bhattacharya et al.
can be found at any given time in the peripheral blood of a
normal mouse, and these cells are capable of reconstituting
irradiated mice and functionally engraft unnirradiated part-
ners in a parabiotic model (16). Blood HSCs have a resi-
dence time in blood of ?1 min, and so to maintain a
steady-state of 100 HSCs, upwards of 30,000 HSCs per day
fl ux through the blood of a mouse (16). Although the etio-
logic reasons are unclear, the presence of these cells in the
blood suggests that HSCs may exist in a dynamic equilib-
rium with their environment and that a certain number of
bone marrow niches may be available for engraftment at
any given point. Second, several recent studies using un-
fractionated bone marrow transplants have shown that the
small congenic diff erences used to distinguish donor-de-
rived hematopoietic cells from the recipient’s endogenous
cells constitute a signifi cant immunologic barrier (17, 18).
Figure 1. Donor HSCs can engraft unconditioned tolerant wild-
type hosts. (A) Persistent multilineage contribution occurs only in
tolerant recipients. 4,000 ckit+ Thy1.1low lineage− Sca-1+ Flk2− CD34−
HSCs from male CD45.1 mice were transplanted into unirradiated male
CD45.1 × CD45.2 (F1), CD45.2, or irradiated male CD45.1 × CD45.2
recipients. Granulocytes were pregated as side scatterhigh B220−
TCRβ− cells, B cells were pregated as Mac-1− TCRβ− cells, and T cells
were pregated as B220− Mac-1− cells. Representative plots of
peripheral blood at 16 wk after transplantation are presented.
(B) Consistent granulocyte contribution in tolerant hosts. Granulocyte
chimerism is shown at 4-wk intervals after transplantation into
CD45.1 × CD45.2 and CD45.2 hosts. ◇, F1 hosts; ●, CD45.2 recipients.
Data points below the dotted line represent animals with no
JEM VOL. 203, January 23, 2006
These studies suggest that the absence of donor-derived
cells in unconditioned hosts receiving congenic hemato-
poietic transplants may be a refl ection of immunologic re-
jection rather than a lack of open HSC niches.
To assess the role of immunosurveillance and to quantify
the number of available HSC niches in normal and immuno-
defi cient animals, we performed a series of HSC transplants
into histocompatible hosts and recipients with a minor histo-
compatibility mismatch at a single locus. We show that trans-
planted HSCs can productively engraft ?0.1–1.0% of HSC
niches in H2-matched, minor histocompatibility– mismatched
unconditioned hosts, but only if host CD4+ T cells are absent
or genetically unreactive to the graft. Large excesses of trans-
planted HSCs do not signifi cantly increase this engraftment,
suggesting that endogenous HSCs residing in appropriate
niches cannot be easily displaced. Despite the small numbers
of available HSC niches, we show that transplantation of pu-
rifi ed HSCs is suffi cient to permanently rescue animals with
severe immunodefi ciencies by regenerating donor-derived
lymphocytes to normal levels through expansion of progeni-
tor cells within the unfi lled lymphoid compartments. The
mechanism by which purifi ed HSC transplantation corrects
lymphoid disorders is likely to be broadly applicable to the
treatment of hematopoietic defi ciencies.
Immune surveillance prevents engraftment of subtly
HSCs in 4,000 ckit+ Thy1.1low lineage− Sca-1+ Flk2−
CD34− cells from CD45.1 mice were transplanted intrave-
nously into fi ve unnirradiated CD45.1 × CD45.2 (F1) and
fi ve unirradiated congenic CD45.2 mice. Every 4 wk after
transplantation, peripheral blood was analyzed for granulo-
cyte, B cell, and T cell chimerism. Donor granulocyte chi-
merism, which accurately refl ects HSC chimerism (16), was
observed only in the genetically unreactive F1 recipients at
all time points analyzed with a median chimerism at 16 wk of
?0.1% (Fig. 1).[ID]FIG1[/ID] These data demonstrate that the subtle anti-
genic diff erences that exist between these CD45 congenic
strains of mice are suffi cient to prevent productive HSC
Rapid HSC-mediated correction of lymphoid defi ciencies in
To gauge the potential clinical importance of these rare avail-
able niches, we repetitively transplanted HSCs from GFP-
transgenic mice into RAG2 and IL-2 receptor common γ
chain–defi cient (RAG2−/−γc−/−) mice, which lack B, T,
and NK cells (19). Enormous numbers of donor-derived B
and T cells were found in the blood at all time points, leading
to an overall donor chimerism of ?50% until at least 30 wk
after the fi nal transplantation (Fig. 2, A and B). [ID]FIG2[/ID] Donor NK
cells were also detected in all transplanted animals (not de-
picted). Moreover, all RAG2−/−γc−/− mice displayed persis-
tent donor-derived myeloid chimerism (ranging from 0.5 to
2.0% donor-derived granulocytes).
To confi rm that the donor granulocyte frequencies ac-
curately refl ected bone marrow HSC chimerism, we killed
animals at 30 wk after transplant and analyzed bone marrow.
Donor cells comprised ?0.8% of the total long-term (LT)-
HSC pool (Fig. 3 A).[ID]FIG3[/ID] This chimerism was essen-tially the
same in the short-term reconstituting stem cells (ckit+ lin-
eage− Sca-1+ CD34+ Flk2−) and the multipotent progeni-
tors (ckit+ lineage− Sca-1+ CD34+ Flk2+; not depicted). We
were unable to detect any cells with these surface pheno-
types in the spleen, a major organ associated with extramed-
ullary hematopoiesis (not depicted). These data confi rmed
that in these animals, peripheral donor granulocyte frequen-
cies much more accurately refl ect HSC chimerism than the
overall donor contribution in the blood. The data also dem-
onstrate that small numbers of bone marrow–engrafted HSCs
can correct severe lymphoid defi ciencies without prior cyto-
reductive conditioning. The HSC chimerism in the RAG2−/−
γc−/− mice was comparable to the chimerism seen in the ge-
netically unreactive F1 wild-type mice (Fig. 1), demonstrat-
ing that there are not obviously greater numbers of available
HSC niches in these animals.
To confi rm that functional HSCs had engrafted, we
isolated bone marrow at 31 wk after transplant from unir-
radiated RAG2−/−γc−/− mice that had received GFP+ HSC
transplants and performed secondary transplants using either
Figure 2. Small numbers of engrafted wild-type HSCs can rescue
lymphocyte defi ciencies in unnirradiated RAG2−/−후c−/− mice.
(A) Robust donor chimerism in unconditioned RAG2−/−γc−/− recipients.
Unirradiated RAG2−/−γc−/− mice were repetitively transplanted six times
with 1,750–4,000 HSCs from eGFP-transgenic mice, and peripheral blood
was analyzed for donor myeloid and lymphoid contribution at 4 wk after
the fi nal transplant. Granulocytes were pregated as side scatterhigh B220−
TCRβ− cells, B cells were pregated as Mac-1− TCRβ− cells, and T cells were
pregated as B220− Mac-1− cells. (B) Persistent multilineage contribution
in unnirradiated RAG2−/−γc−/− recipients. Peripheral blood was analyzed
for donor contribution periodically until 30 wk after fi nal transplantation.
76 ENGRAFTMENT IN RARE EMPTY HEMATOPOIETIC STEM CELL NICHES | Bhattacharya et al.
unfractionated or c-kit–enriched marrow, which increases
the frequency of HSCs by ?10-fold, into lethally irradiated
wild-type mice, such that ?6–20 GFP+ HSCs along with
1,000 RAG2−/−γc−/− HSCs were transferred into each
secondary recipient. Donor-derived GFP+ cells were ob-
served in all secondary recipients until at least 25 wk after
transplantation, and 6 out of 13 secondary recipients main-
tained detectable levels of granulocyte chimerism (Fig.
3 B). These data confi rmed that rare GFP+ HSCs within
the bone marrow of the primary RAG2−/−γc−/− recipients
had productively engrafted. In contrast, transplantation of
large numbers of splenocytes into secondary recipients did
not lead to sustained multilineage stem cell reconstitution
Opportunistic expansion of donor lymphoid progenitors
To determine the developmental stage at which donor B cells
overtake host B cells in RAG2−/−γc−/− recipients, we ana-
lyzed donor frequencies in myeloid and lymphoid progenitor
cells in the bone marrow. Common myeloid progenitors
(CMPs; reference 20) and common lymphoid progenitors
(CLPs; references 21–23) showed donor chimerism that was
comparable to HSC chimerism (Fig. 4), indicating that do-
nor-derived cells do not have a competitive proliferative ad-
vantage at these early developmental steps.[ID]FIG4[/ID] Donor chimerism
at the granulocyte macrophage progenitor (GMP) and mega-
karyocyte erythrocyte progenitor (MEP) developmental steps
were also similar to HSC chimerism (not depicted). Consis-
tent with these results, the frequencies of endogenous HSCs,
CLPs, CMPs, GMPs, and MEPs within the bone marrow are
similar between untransplanted wild-type and RAG2−/−
γc−/− mice (Fig. 5).[ID]FIG5[/ID] Analysis of the pro–B-A and pro–B-B
cell fractions (24, 25), however, showed donor chimerism
that was dramatically higher than the preceding CLP (Fig. 4).
At the pro–B-B cell stage and all subsequent B cell stages,
cells were exclusively donor-derived. Although IL-7 recep-
tor, which uses γc for proper signaling, is expressed at the
CLP stage, these results suggest that IL-7 signaling is not a
Figure 3. Low-level HSC engraftment in primary unconditioned
RAG2−/−후c−/− recipients. (A) Donor HSCs can be detected in primary
unirradiated RAG2−/−γc−/− recipients. At 31 wk after the fi nal trans-
plantation, donor HSC contribution in the bone marrow in uncondi-
tioned primary RAG2−/−γc−/− recipients was quantifi ed using the
markers shown. (B) Engrafted HSCs in the bone marrow of uncondi-
tioned primary RAG2−/−γc−/− recipients can be serially transplanted.
Unfractionated (5 × 107) or c-kit–enriched (106) bone marrow cells
from primary RAG2−/−γc−/− recipients were secondarily transplanted
into lethally irradiated (950 cGy) wild-type mice. GFP+ chimerism
was assessed at 25 wk after secondary transplantation. Each second-
ary recipient received a transplant from a distinct nonredundant
primary donor. Data points below the dotted line represent undetect-
JEM VOL. 203, January 23, 2006
requisite pathway for CLP development or expansion, con-
sistent with previous observations (26).
Normal immune responses in HSC-reconstituted SCID mice
To verify that the immune system of the HSC-reconstituted
RAG2−/−γc−/− mice had been restored and was capable of
mounting appropriate immune responses, we immunized re-
constituted recipients with alum-precipitated 4-hydroxy-3-
nitrophenylacetyl (NP) conjugated to chicken γ globulin,
which elicits a Th2-dependent humoral response (27, 28).
Serum levels of NP-specifi c antibody were similar at 1 wk after
immunization between HSC-transplanted RAG2−/−γc−/−
and wild-type mice, demonstrating the immunocompetence
of the transplanted RAG2−/−γc−/− recipients (Fig. 6).[ID]FIG6[/ID]
Host CD4+ T cells are essential for rejection of subtly
To investigate whether the absence of γc was critical for
HSC engraftment in unconditioned hosts, perhaps by im-
parting a competitive disadvantage on HSCs in RAG2−/−
γc−/− animals, we transplanted GFP+ HSCs into RAG2−/−
mice, which have normal γc expression, as well as into
RAG2−/−γc−/− mice. As shown in Fig. 7, similar levels of
donor granulocyte contribution were seen in RAG2−/− and
RAG2−/−γc−/− mice, likely excluding a direct role for γc in
maintaining host HSCs within their niches. [ID]FIG7[/ID] However, γc ex-
pression has been observed in normal HSCs (29), suggesting
that there may be a slight competitive advantage for HSCs
with proper γc expression. The data also suggest that NK
cells, which are present in normal numbers in RAG2−/−
mice but absent in RAG2−/−γc−/− mice, are not mediating
HSC rejection in this H2-identical system. Elimination of
host NK cells is required for engraftment of HSCs that carry
one or more unshared H2 haplotype (30). Interestingly, do-
nor-derived lymphocyte frequencies were signifi cantly re-
duced in RAG2−/− recipients at early time points (Fig. 7 B),
perhaps as a result of the occupation of the lymphoid stage–
specifi c stromal environments by RAG2−/− lymphoid pro-
genitors (31). The number of donor-derived B cells in
RAG2−/− recipients was reduced more than 10-fold relative
to RAG2−/−γc−/− recipients at 4 wk after transplant, and
peripheral T cells were not seen at all until 8 wk after trans-
plant (not depicted). However, by 16 wk after transplanta-
tion, B and T cell numbers in RAG2−/− recipients reached
the levels seen in their RAG2−/−γc−/− counterparts (Figs.
7 B and 8 A).[ID]FIG8[/ID]
Because RAG2−/− mice lack both mature B and T cells,
we sought to determine which of these cell types was primar-
ily responsible for mediating the rejection of transplanted do-
nor HSC grafts. Therefore, we transplanted purifi ed HSCs
from GFP donor mice into unnirradiated TCRα−/−β−/−
and Cμ−/− mice. In these experiments, known antigenic
diff erences between donor HSCs and recipient mice exist at
the GFP, CD45, and Thy1 loci. Multilineage engraftment
was observed in T cell–defi cient mice, but not in B cell–
defi cient mice (Fig. 8 A). These experiments show that host
αβ T cells are required for the rejection of HSC grafts with
these minor histocompatibility mismatches. To determine
which class of T cells is essential for this immunosurveillance,
we transplanted HSCs into unconditioned I-A−/− mice,
which lack MHC II–restricted CD4+ T cells (32), and β2-
microglobulin−/− (β2m−/−) mice, which are defi cient in
MHC I–restricted CD8+ T cells (33). The HSC-transplanted
Figure 5. Stem and progenitor cell frequencies are normal in
RAG2−/−후c−/− mice. LT-HSCs, ST-HSCs, and multipotent progenitors
were stained from untransplanted mice as in Fig. 2. CLPs and CMPs were
stained as in Fig. 4. MEPs and GMPs were stained according to established
protocols (reference 20). Each endogenous population is presented as a
percentage of total bone marrow. ○, wild-type animals; ◇, RAG2−/−
Figure 6. Reconstituted RAG2−/−후c−/− mice can respond normally
to antigenic challenge. RAG2−/−γc−/− mice were immunized with 100
μg alum-precipitated NP–chicken γ globulin 16 wk after HSC transplant.
Serum was obtained 1 wk after immunization and tested for NP-specifi c
antibodies. Mice with undetectable serum levels of NP-specifi c IgG are
shown below the dotted line.
Figure 4. Signifi cant donor cell expansion is not observed until
the pro–B cell stage. At 31 wk after the fi nal transplantation, donor
contribution to myeloid and lymphoid progenitor cells was analyzed.
CMPs were defi ned as lin− ckit+ Sca-1− CD16/32low CD34low cells. CLPs
were gated as lin− Thy1.1low IL7Rα+ Flk2+ ckitlow Sca1low cells. Pro–B-A
cells were defi ned as B220+ CD43+ IgM− CD19− NK1.1−, and pro–B-B
cells were gated as B220+ CD43+ IgM− CD19+ Ly51− NK1.1− cells. Mean
values ± SEM are shown from the analysis of three mice for each subset.
78 ENGRAFTMENT IN RARE EMPTY HEMATOPOIETIC STEM CELL NICHES | Bhattacharya et al.
I-A−/− mice showed sustained chimerism until at least 16
wk, whereas the β2m−/− recipient mice did not show chime-
rism at any time point (Fig. 8 B), thereby demonstrating that
CD4+ T cells are essential for the rejection of minor histo-
compatibility–mismatched HSC grafts in our system. LT-
HSCs express MHC II (Fig. 8 C) and the costimulatory
molecule CD86 (29), suggesting that host CD4+ T cells may
directly recognize HSCs with slight antigenic mismatches.
Although it appears that CD8+ T cells are not required for
this rejection, we cannot exclude the possibility that residual
hyperreactive MHC I–restricted T cells might also contribute
to HSC graft rejection in the β2m−/− mice (34–36). Consis-
tent with the role for CD8 T cells in mediating bone marrow
graft rejection, Xu et al. (17) have shown that host CD8 de-
fi ciency enhances engraftment.
Interestingly, the granulocyte chimerism in the RAG2−/−
recipients was indistinguishable from that seen in previous
experiments in which 3,000 HSCs rather than 1,000 were
transplanted (Fig. 7). In contrast, although transplantation of
20 HSCs led to detectable B and T cell production in these
immunodefi cient mice, granulocyte chimerism was barely
detectable (not depicted). Thus, our experiments suggest that
HSC engraftment and chimerism asymptotically approaches a
maximum of ?0.5% in a cell dose–dependent manner. The
data show that in contrast to previous speculations, endoge-
nous HSCs cannot be displaced from the niches they occupy
by increasing transplanted HSC numbers above a threshold
level. In repetitively transplanted mice, however, we have
observed small increases in granulocyte chimerism relative to
mice that were HSC-transplanted only once with doses above
this threshold (Fig. 2 B vs. Fig. 7 B). This provides evidence
that transplantation of an excess of HSCs does not preclude
additional niches from being vacated in the future.
To determine if transient CD4+ T cell removal would
allow access of transplanted HSCs to appropriate niches, we
treated Cμ−/− mice with a depleting CD4 antibody that led
to ?95% depletion of peripheral blood CD4+ T cells (Fig. 9
A).[ID]FIG9[/ID] CD4-depleted mice were then transplanted with 800
HSCs and analyzed at various time points for donor chime-
rism. All mice that received anti-CD4 treatment showed
Figure 7. 후c expression is not directly important for HSC mainte-
nance. (A) Purifi ed wild-type HSCs can engraft unirradiated RAG2−/−
mice. Unconditioned RAG2−/− and RAG2−/−γc−/− mice were transplanted
with 3,000 HSCs from GFP-transgenic mice. Representative plots from
peripheral blood obtained 4 wk after transplantation are shown. Granulo-
cytes were pregated as side scatterhigh B220− CD3− cells, B cells were
pregated as Mac-1− CD3− cells, and T cells were pregated as B220−
Mac-1− cells. (B) Persistent granulocyte chimerism but delayed lymphoid
reconstitution in RAG2−/− mice. ◇, RAG2−/−γc−/− animals;
●, RAG2−/− animals.
JEM VOL. 203, January 23, 2006
donor granulocyte chimerism, whereas none of the untreated
mice displayed any detectable donor cells at 8 wk after trans-
plantation (Fig. 9 B). Signifi cant numbers of donor B cells
were observed in the treated mice at 6–8 wk after transplan-
tation, again demonstrating the ability of small numbers of
productively engrafted HSCs to restore lymphocyte numbers
in immunodefi cient mice. Because host CD4+ T cells levels
were noted to recover subsequent to immunodepletion (not
depicted), these experiments demonstrate that transient de-
pletion of these cells before transplantation is suffi cient to al-
low for productive short-term stem cell engraftment without
the need for the standard toxic cytoreductive drugs com-
monly used for B(-) non-SCID patients before bone marrow
transplant (37). However, by 12 wk after HSC transplanta-
tion, donor B cell, T cell, and myeloid chimerism was lost in
all recipient mice (not depicted). These data suggest that ei-
ther the α-CD4–mediated depletion of mature T cells was
incomplete, or that suffi cient numbers of donor-derived den-
dritic cells were not generated to mediate lasting tolerance
through negative selection in the thymus (38).
Short-term reconstitution in unconditioned aged recipients
Aged individuals show marked reductions in thymus size
and T cell function (39). To determine if the reduced lym-
phoid function in aged mice would allow acceptance of
transplants without conditioning, we repetitively trans-
planted HSCs from GFP-transgenic mice into old (22 mo)
and young (2 mo) recipients. As seen in Fig. 10, short-term
low-level myeloid chimerism was observed in all old re-
cipients in contrast to the young recipients that showed
to undetectable levels in all but one of the old recipients
with time, suggesting that rejection of the transplants did
occur, but with signifi cantly reduced kinetics relative to
the younger animals (not depicted). The level of granulo-
cyte chimerism in this experiment was similar to the low
levels seen in transplants of younger, genetically unreactive
or immunodefi cient mice, suggesting that aged HSCs can-
not be displaced from their endogenous niches. This is in
contrast to previous studies performed with unfractionated
bone marrow transplants (40) and suggests that reduced
[ID]FIG10[/ID] However, donor-derived cells declined
Figure 8. Host T cells act as barriers to productive HSC engraft-
ment. (A) α/β T cells are required for HSC graft rejection. 1,000 puri-
fi ed HSCs from eGFP-transgenic mice were transplanted into
TCRα−/−β−/−, Cμ−/−, RAG2−/−, and wild-type animals. Peripheral
blood was analyzed for donor cell contribution at 16 wk after trans-
plantation. Granulocytes were pregated as side scatterhigh B220−
TCRβ− cells, B cells were pregated as Mac-1− TCRβ− cells, and T cells
were pregated as B220− Mac-1− cells. A representative plot for each
group is shown. (B) Host CD4+ T cells are essential for HSC graft rejec-
tion. 800 purifi ed HSCs from GFP-transgenic mice were transplanted
into 2 I-A−/− and 3 β2m−/− mice. Peripheral blood was analyzed 16 wk
after transplantation for donor chimerism. Representative plots are
shown. (C) LT-HSCs express MHC II. MHC II expression was analyzed on
LT-HSCs from wild-type animals.
80 ENGRAFTMENT IN RARE EMPTY HEMATOPOIETIC STEM CELL NICHES | Bhattacharya et al.
immune capacity is responsible for short-term engraftment
of donor HSCs.
The remarkable ability of HSCs to sustain multilineage he-
matopoiesis for the lifetime of an individual constitutes the
foundation for their routine use in a range of clinical appli-
cations, including the treatment of primary immunodefi -
ciencies (41, 42), malignancies (43–45), as conditioners for
transplantation tolerance of tissue or organ grafts from the
donors (46), and as a method to reverse some types of auto-
immunity (47). The success of such therapies relies on the
ability of HSCs to home to unique niches leading to sus-
tained multilineage hematopoiesis. The studies presented
here have quantifi ed the number of these HSC niches that
are available for engraftment at any given point in uncondi-
tioned animals as ?0.1–1.0% of all HSC niches. Assuming a
total adult murine bone marrow cellularity of 5 × 108 cells
(48) and an endogenous HSC frequency of 0.01% (Fig. 4),
the number of open HSC niches can be estimated to be 50–
500. This is strikingly similar to the number of HSCs esti-
mated to be in circulation at any given point (16). The data
suggest that HSCs that circulate normally have exited and left
vacant their previous HSC niche. Thus, a constant exchange
may be occurring between endogenous HSCs under normal
circumstances, perhaps to maintain hematopoietic balance
between and within each bone marrow compartment. In sup-
port of this, we have found little diff erence in the granulocyte
Figure 9. Transient CD4 depletion allows productive engraft-
ment of HSCs with minor histocompatibility mismatches. (A) Treat-
ment of Cμ−/− mice with a depleting antibody leads to effi cient CD4+
T cell removal. Mice were treated consecutively for 3 d with anti-CD4
antibody, and peripheral blood was analyzed for TCRβ+ CD8− cells to
assess depletion 1 d after the fi nal treatment. The plots are gated on
Ter119− TCRβ+ cells. (B) HSC engraftment occurs in anti-CD4–treated
animals. Three Cμ−/− mice that were treated with anti-CD4 antibody
and two Cμ−/− mice that were left untreated were transplanted with
800 GFP+ HSCs. Peripheral blood was analyzed at 8 wk after transplan-
tation for granulocyte, B cell, and T cell chimerism. Granulocytes
were pregated as side scatterhigh B220− CD3− cells, B cells were pre-
gated as Mac-1− CD3− cells, and T cells were pregated as B220− Mac-1−
cells. Representative plots are shown. (C) Signifi cant short-term multi-
lineage engraftment in all anti-CD4–treated recipients. Donor con-
tribution to granulocytes, T cells, and B cells at 8 wk after HSC
transplant is shown. B cells are quantifi ed as number of cells contained
within 100 μl of peripheral blood. ◇, anti-CD4–treated animals;
●, untreated animals. Data points that were undetectable are shown
below the dotted line.
JEM VOL. 203, January 23, 2006
chimerism rates between experiments when a single trans-
plant of HSCs is provided in doses ranging from 800 to 4,000
cells. Although previous reports have suggested that the cell
doses of transplanted bone marrow correlate with total chi-
merism linearly, such data at most show replacement of bone
marrow and mature cells in bulk and do not refl ect replace-
ment of HSCs, which represent only 0.01% of unfractionated
marrow (9). When HSCs are repetitively transplanted, how-
ever, we have observed increases in granulocyte chimerism
(Fig. 2 B vs. Fig. 7 B). Thus, occupation of available HSC
niches after transplantation of an excess of exogenous HSCs,
which remain in circulation for ?1–5 min after transplanta-
tion (16), does not preclude additional niches from becoming
available subsequently. Conceivably, continuous transfusion
of low numbers of HSCs would be superior to singly admin-
istered boluses, as the rate of niche emptying and fi lling is
high. Because there does not appear to be an obvious increase
in granulocyte chimerism with time or cell dose above a
threshold level, the data also suggest that transplanted HSCs
must fi nd their way rapidly to an appropriate niche and can-
not recirculate indefi nitely in search of empty niches without
the loss of hematopoietic potential.
The ability of transplanted HSCs to self-renew for the
lifetime of the organism ensures a constant production of
normal lymphoid cells through each developmental stage. In
the genetic mutants used in our work, host lymphocyte de-
velopment is blocked or perturbed at defi ned developmental
stages. At each developmental stage or thereafter, wild-type
donor cells have a competitive advantage and can opportu-
nistically expand or accumulate to ultimately give rise to large
numbers of normal mature lymphocytes. Several factors have
been implicated in the expansion of the early B cell and thy-
mocyte lineages, including IL-7 (24, 49), stem cell factor
(50), Flt3 ligand (22, 51), and recently, various Wnt/Frizzled
pathways (52). In the case of γc−/− animals, the pro–B-B
population appears to have defects in IL-7–dependent expan-
sion, providing a proliferative advantage to wild-type donor
cells at these stages (24, 53–56). RAG2−/− mice likely recon-
stitute more slowly because their lymphocytes can develop
normally through the pro–B cell as well as DN3 thymocyte
stages and occupy the appropriate stromal microenviron-
ments (31). However, because RAG2−/− lymphocytes can-
not advance past these stages (57), small numbers of developing
donor-derived cells can expand and accumulate without
competition at the pre–B as well as DN4 thymocyte cell
stages and all subsequent developmental steps. Although it is
possible that the donor LT-HSCs will only persist for fi nite
periods of time because of the considerable demand imposed
by the ?250,000-fold expansion to the lymphocyte stage, we
have observed no meaningful decline in granulocyte or lym-
phocyte chimerism at any time point up to 30 wk after trans-
plantation of primary recipients. Nonetheless, because we
have observed some declines in HSC potency after secondary
transplantation (Fig. 3 B vs. Fig. 2 B), in clinical settings it
would be advisable to keep donor LT-HSCs stored in the
event that grafts do not persist indefi nitely. Additionally, the
low levels of granulocyte chimerism achieved from a single
transplant are unlikely to be clinically useful for patients suf-
fering from myeloid defi ciencies.
We also show conclusively that stable engraftment within
these rare niches by minor histocompatibility–mismatched
HSCs is tightly regulated by host CD4+ T cells. HSCs from
CD45.1 mice cannot productively engraft unirradiated con-
genic CD45.2 mice, yet they routinely engraft the genetically
unreactive F1 strain (CD45.1 × CD45.2). To our knowl-
edge, the only antigenic diff erence between these strains is
the CD45 allele, which is normally considered to be a rela-
tively innocuous congenic marker. Similarly, HSCs isolated
from GFP-transgenic mice backcrossed to the C57BL/Ka
genetic background cannot productively engraft wild-type
C57BL/Ka mice. The only antigenic diff erence between
these strains to our knowledge is the GFP gene product.
These experiments prove that very small antigenic diff erences
lead to a complete rejection of donor HSC grafts in the ab-
sence of cytoreductive conditioning.
Encouragingly, however, the elimination of CD4+ T cell
function allows for the functional and sustained engraftment of
HSCs with minor histocompatibility mismatches in our system.
Xu et al. (17) have shown that transient antibody-mediated
depletion of host αβ T cells in mice enhances engraftment
of donor bone marrow with minor histocompatibility mis-
matches. Spitzer et al. (58) have shown that conditioning hap-
loidentical patients with a depleting α-CD2 antibody along
with low-dose cytoreductive treatments before bone marrow
transplantation allows, at the minimum, transient multilineage
engraftment. Consistent with this, we demonstrate that tran-
sient antibody-mediated CD4+ T cell depletion alone is suf-
fi cient to allow short-term engraftment of wild-type donor
HSCs and restoration of B cells in a mouse model of non-
SCID. More complete CD4+ T cell depletions and/or better
methods to increase donor-derived thymic dendritic cell con-
tribution might allow for lasting donor hematopoiesis.
Even in the absence of inherited genetic mutations, both
mice and humans develop diminished immune capacity with
age. This progressive loss of immune function has recently
Figure 10. Transplanted HSC grafts are accepted more readily in
aged recipients than in young mice. Purifi ed eGFP+ HSCs were sorted
and 1,750–4,000 cells were repetitively transplanted into 2-mo-old
C57Bl6/Ka and 24-mo-old C57Bl6/Harland mice. Donor granulocyte, B
cell, and T cell chimerism are shown from the fi rst peripheral bleed taken
at 6.5 wk after the fi rst transplant (3 d after the fi nal transplant).
●, young recipients; ◇, aged recipients. The numbers of animals with no
detectable chimerism in each group are listed below the dotted line.
82 ENGRAFTMENT IN RARE EMPTY HEMATOPOIETIC STEM CELL NICHES | Bhattacharya et al.
been attributed to HSC-intrinsic defects in diff erentiation to
lymphoid-primed progenitors (29). Because we have dem-
onstrated that a very small number of properly functioning
HSCs can mask the defects in a much larger pool of HSCs, it
is tempting to speculate that age-related immune defects
don’t become readily apparent until nearly all fully “young”
HSCs are exhausted. The reintroduction of fully multipotent
HSCs, perhaps obtained as an autologous sample earlier in
life, might signifi cantly delay age-related immune decline.
Numerous studies have shown how common condition-
ing treatments used before bone marrow transplantation,
such as irradiation, cyclophosphamide, and busulfan, can
cause serious side eff ects, including lowered platelet counts,
infertility, and secondary malignancies (59). When these cy-
totoxic therapies are used to treat hematologic malignancies,
the side eff ects must unfortunately be tolerated as a byproduct
of necessary chemotherapy. The necessity of such condition-
ing treatments for hematopoietic defi ciencies before HSC
transplantation, however, should be reconsidered. Although
it is true that available niche space is low under normal con-
ditions, we show that transplantation of modest numbers of
highly purifi ed HSCs can engraft the few niches that are
available and correct lymphoid defi ciencies. Thus, niche
space is not an absolute limiting factor to HSC-mediated cor-
rection of B, T, or NK cell defi ciencies.
Unlike the myeloablative regimens almost always per-
formed on non-SCID immunodefi cient patients, SCID pa-
tients who receive MHC-matched CD34-enriched or T
cell–depleted bone marrow grafts generally do not receive
cytoreductive conditioning before transplantation (37, 60).
However, it has been suggested that HSC engraftment does
not occur in these patients (61). Because many of these pa-
tients show poor B cell lymphopoiesis and lose T cell counts
with time, it has been proposed that the lymphoid correction
occurs as a result of engraftment of short-lived progenitor
cells, which along with mature cells constitute the vast ma-
jority of transplanted cells, rather than HSCs with full he-
matopoietic and self-renewal potential (62, 63). A careful
examination of the data, however, shows that ?0.8% of
CD34+ cells in an unconditioned SCID patient who received
a bone marrow transplant are not of host origin (61). Al-
though the authors, understandably, did not consider this
level of engraftment meaningful, our results suggest that small
numbers of HSCs have engrafted in these patients and that
eventual T cell loss may be a refl ection of HSC exhaustion
rather than an initial failure to engraft. This hypothesis is re-
inforced by suggestions that the process of physiological HSC
circulation seems to be conserved between mice and humans
(16, 64, 65). Alternatively, the lack of B and T lymphopoiesis
has correlated well with graft-versus-host disease (GVHD) in
previous studies (66, 67). In MHC-matched settings, bone
marrow grafts are often transplanted without manipulation,
whereas in HLA-mismatched settings, T cell depletions or
CD34 enrichments of donor marrow can still leave up to 104
T cells/kg (37). Our results show that even in MHC-matched
congenic mouse model systems, immune responses can rec-
ognize and reject very slightly mismatched cells, suggesting
that it is very likely that GVH responses occur in all patients
that receive any mature T cells as part of their nonautologous
graft. Although GVHD may not be classifi ed as clinically sig-
nifi cant or obviously symptomatic in all cases, subtle GVH
eff ects on B and T lymphopoiesis might still occur. Thus, the
use of purifi ed HSC transplants, which do not cause GVHD
(30), may potentially avoid poor B lymphopoiesis. In our
mouse model system, we have observed sustained B and T
lymphopoiesis for the duration of our experiments after puri-
fi ed HSC transplantation.
The mechanism by which transplanted HSCs correct
hematopoietic defi ciencies in our unconditioned recipients is
applicable to the correction of many types of both SCID and
non-SCID immunodefi ciencies, but these studies at the same
time clearly demonstrate that very subtle minor histocompat-
ibility diff erences can mediate the rejection of HSC grafts
when host T lymphocytes are present. Our data suggest that
transplantation of purifi ed HSCs, in combination with highly
specifi c lymphoablative treatments when necessary, can
correct lymphoid defi ciencies in immunodefi cient patients
without the undesired side eff ects, such as toxic conditioning
and GVHD, often associated with current conditioning and
transplantation regimens. Future experiments will determine
if the same strategy can be applied to the correction of my-
eloid defi ciencies.
MATERIALS AND METHODS
Animals. All animal procedures were approved by the International Animal
Care and Use Committee. C57BL/Ka-Thy1.1 CD45.2+ (HZ) and C57BL/
Ka-Thy1.1 CD45.1 (BA) strains were derived and maintained in our labora-
tory. eGFP transgenic mice used in these studies were backcrossed at least 20
generations to either the BA or HZ strain. C57Bl6/Harland mice used for
the aging studies were obtained from the National Institute of Aging. The
RAG2−/−, RAG2−/−γc−/−, I-A−/−, and β2m−/− mice have been described
previously (19, 32, 36, 57) and were bred at least 20 generations onto the
C57BL/Ka-Thy1.2 CD45.1+, C57BL/Ka-Thy1.2 CD45.2+, and C57BL/
Ka-Thy1.1 CD45.2 backgrounds. TCRα−/−β−/− mice were provided by
J. Campbell and M. Davis (Stanford University, Stanford, CA). Cμ−/− mice
were provided by J. Tung and L. Herzenberg (Stanford University). Periph-
eral blood was sampled from the tail vein, and all HSC transplants were per-
formed by injection into the retroorbital sinus of isofl urane-anesthetized
mice. For repetitive transplants, between 1,750 and 4,000 HSCs were trans-
planted weekly for 6 wk. Donor mice were 4–6-wk old, and recipient mice
ranged from 4–12 wk of age unless otherwise noted.
Antibodies. The following monoclonal antibodies were purifi ed and con-
jugated using hybridomas maintained in our laboratory: 19XE5 (anti-
Thy1.1), 2C11 (anti-CD3), GK1.5 (anti-CD4), 53-7.3 (anti-CD5), 53-6.7
(anti-CD8), 6B2 (anti-B220), 8C5 (anti–Gr-1), M1/70 (anti–Mac-1),
TER119 (anti-Ter119), A20.1.7 (anti-CD45.1), AL1-4A2 (anti-CD45.2),
2B8 (anti–c-kit), E13-161-7 (anti–Sca-1), anti-CD16/CD32 (2.4G2), and
A7R34 (anti–IL-7 receptor α). Antibodies were conjugated to biotin, PE,
allophycocyanin (APC), Alexa 405, Alexa 430, or Alexa 488 (Invitrogen),
according to the manufacturer’s instructions. The following were purchased
from eBiosciences: antibodies against CD3, CD4, CD8, B220, Mac-1,
Ter119, and Gr-1 conjugated to PE-Cy5; anti–c-kit and anti–Mac-1 conju-
gated to PE-Cy7; anti–Sca-1 conjugated to PE-Cy5.5; anti-CD45.1 and
anti-CD45.2 (104) conjugated to APC-Cy5.5; anti-B220 conjugated to
APC-Cy7; anti-Flk2 (A2F10) conjugated to PE or biotin; and streptavidin
conjugated to APC. Anti-CD34 (RAM34) conjugated to FITC or biotin,
JEM VOL. 203, January 23, 2006
anti-TCRβ (H57-597) conjugated to APC, anti-CD43 (1B11) conjugated
to PE, anti–I-A/I-E (M22.214.171.124) conjugated to PE, and anti-Ly51 (6C3)
conjugated to biotin were purchased from BD Biosciences. Streptavidin
conjugated to APC-Cy7 was purchased from Caltag.
FACS and analysis. All cells were sorted on a FACSVantage or a FACS Aria
(Becton Dickinson). All peripheral blood analysis was performed on an LSR-
Scan (Becton Dickinson). Peripheral blood was obtained from the tail vein, red
blood cells were sedimented with 2% dextran, and the remaining red blood
cells were lysed with an ammonium chloride solution. The remaining white
blood cells were stained with anti-CD45.2–Alexa 488, anti–CD45.1-PE, anti–
Ter119-PE-Cy5, anti–Mac-1–PE-Cy7, anti–TCRβ-APC, and anti–B220-
APC–Cy7. When peripheral blood containing eGFP+ cells was analyzed,
anti-CD45.1–Alexa-488 was omitted and anti–Gr-1–PE was used instead of
anti–CD45.2-PE if warranted by the experiment. For HSC isolation, bone
marrow was fi rst enriched using anti–c-kit beads and immunomagnetic selec-
tion was performed on an AutoMACS machine (Miltenyi Biotec). Enriched
cells were stained with anti–CD34-FITC, anti–Flk2-PE, anti-lineage (CD3,
CD4, CD8, B220, Ter119, Mac-1, Gr-1)–PE-Cy5, anti–Sca-1–PE-Cy5.5,
and anti–c-kit–PE-Cy7, and cells were double sorted before transplantation.
For analysis of HSC chimerism, CD45.1–APC-Cy5.5 was included, and
CD34 staining was achieved with anti-CD34 biotin followed by streptavidin
APC. For isolation of HSCs from eGFP-transgenic mice, cells were stained
with anti-lineage–Cy5-PE, anti–Sca-1–PE-Cy5.5, anti–c-kit–PE-Cy7, anti–
Flk2-PE, and anti-CD34 biotin followed by streptavidin APC. For analysis of
eGFP HSC chimerism, anti–CD45.1-APC–Cy5.5 was included.
Immunizations. Mice were immunized intraperitoneally with 100 μg NP
conjugated to chicken γ globulin (Biosearch Technologies) precipitated in
10% aluminum potassium sulfate (Sigma-Aldrich). NP-specifi c enzyme-
linked immunosorbent assays were performed with serum obtained 1 wk af-
ter immunization on high-protein binding 96-well plates coated with 5 μg
NP-BSA (Biosearch Technologies). Wells were developed with anti–mouse
IgG-horseradish peroxidase (Southern Biotechnology Associates, Inc.) fol-
lowed by 1 mg/ml ABTS reagent (Sigma-Aldrich), and the reactions were
stopped by the addition of 0.1% sodium azide (Sigma-Aldrich). Absorbance
was read at a wavelength of 405 nm.
In vivo depletion of CD4+ T cells. Mice were treated consecutively for
3 d with 500 μg of intravenously injected purifi ed anti-CD4 antibody
(GK1.5). Peripheral blood was analyzed for TCRβ and CD8 expression to
quantitatively assess CD4+ T cell depletion relative to untreated animals.
These mice were then transplanted with 800 GFP+ HSCs 1 d after the third
injection. The mice were then given weekly injections of anti-CD4 for the
fi rst 3 wk after HSC transplantation and left untreated afterward.
We thank L. Jerabek for laboratory management, C. Richter for antibody production,
L. Hidalgo for animal care, and M. Davis and L. Herzenberg for providing mice.
This work was supported by National Institutes of Health grants
5R01HL058770 and 2R01AI047457 (to I.L. Weissman). D. Bhattacharya is supported
by a fellowship from the Cancer Research Institute, D.J. Rossi is supported by a
fellowship from the Damon Runyon Cancer Foundation, and D. Bryder is supported
by a scholarship from the Swedish Medical Research Council.
Affi liations that might be perceived to have biased this work are as follows: I.L.
Weissman was a member of the scientifi c advisory board of Amgen and owns
signifi cant Amgen stock, cofounded and consulted for Systemix, is a cofounder and
director of Stem Cells, Inc., and recently cofounded Cellerant, Inc. All other authors
have no confl icting fi nancial interests.
Submitted: 24 August 2005
Accepted: 21 November 2005
1. Morrison, S.J., A.M. Wandycz, K. Akashi, A. Globerson, and I.L.
Weissman. 1996. The aging of hematopoietic stem cells. Nat. Med. 2:
2. Schofi eld, R. 1978. The relationship between the spleen colony-
forming cell and the haemopoietic stem cell. Blood Cells. 4:7–25.
3. Hackney, J.A., P. Charbord, B.P. Brunk, C.J. Stoeckert, I.R. Lemischka,
and K.A. Moore. 2002. A molecular profi le of a hematopoietic stem cell
niche. Proc. Natl. Acad. Sci. USA. 99:13061–13066.
4. Zhang, C.C., and H.F. Lodish. 2004. Insulin-like growth factor 2 ex-
pressed in a novel fetal liver cell population is a growth factor for hema-
topoietic stem cells. Blood. 103:2513–2521.
5. Calvi, L.M., G.B. Adams, K.W. Weibrecht, J.M. Weber, D.P. Olson,
M.C. Knight, R.P. Martin, E. Schipani, P. Divieti, F.R. Bringhurst,
et al. 2003. Osteoblastic cells regulate the haematopoietic stem cell
niche. Nature. 425:841–846.
6. Zhang, J., C. Niu, L. Ye, H. Huang, X. He, W.G. Tong, J. Ross, J.
Haug, T. Johnson, J.Q. Feng, et al. 2003. Identifi cation of the haema-
topoietic stem cell niche and control of the niche size. Nature. 425:
7. Osawa, M., K. Hanada, H. Hamada, and H. Nakauchi. 1996. Long-
term lymphohematopoietic reconstitution by a single CD34-low/nega-
tive hematopoietic stem cell. Science. 273:242–245.
8. Wagers, A.J., R.I. Sherwood, J.L. Christensen, and I.L. Weissman.
2002. Little evidence for developmental plasticity of adult hematopoi-
etic stem cells. Science. 297:2256–2259.
9. Rao, S.S., S.O. Peters, R.B. Crittenden, F.M. Stewart, H.S. Ramshaw,
and P.J. Quesenberry. 1997. Stem cell transplantation in the normal
nonmyeloablated host: relationship between cell dose, schedule, and en-
graftment. Exp. Hematol. 25:114–121.
10. Porritt, H.E., K. Gordon, and H.T. Petrie. 2003. Kinetics of steady-
state diff erentiation and mapping of intrathymic-signaling environments
by stem cell transplantation in nonirradiated mice. J. Exp. Med. 198:
11. Ron, Y., and J. Sprent. 1985. Prolonged survival in vivo of unprimed
B cells responsive to a T-independent antigen. J. Exp. Med. 161:
12. Adolfsson, J., O.J. Borge, D. Bryder, K. Theilgaard-Monch, I. Astrand-
Grundstrom, E. Sitnicka, Y. Sasaki, and S.E. Jacobsen. 2001. Upregulation
of Flt3 expression within the bone marrow Lin(−)Sca1(+)c-kit(+)
stem cell compartment is accompanied by loss of self-renewal capacity.
13. Forster, I., and K. Rajewsky. 1990. The bulk of the peripheral B-cell
pool in mice is stable and not rapidly renewed from the bone marrow.
Proc. Natl. Acad. Sci. USA. 87:4781–4784.
14. Tarbell, N.J., D.A. Amato, J.D. Down, P. Mauch, and S. Hellman.
1987. Fractionation and dose rate eff ects in mice: a model for bone
marrow transplantation in man. Int. J. Radiat. Oncol. Biol. Phys. 13:
15. Tomita, Y., D.H. Sachs, and M. Sykes. 1994. Myelosuppressive con-
ditioning is required to achieve engraftment of pluripotent stem cells
contained in moderate doses of syngeneic bone marrow. Blood. 83:
16. Wright, D.E., A.J. Wagers, A.P. Gulati, F.L. Johnson, and I.L.
Weissman. 2001. Physiological migration of hematopoietic stem and
progenitor cells. Science. 294:1933–1936.
17. Xu, H., B.G. Exner, P.M. Chilton, C. Schanie, and S.T. Ildstad. 2004.
CD45 congenic bone marrow transplantation: evidence for T cell-
mediated immunity. Stem Cells. 22:1039–1048.
18. van Os, R., T.M. Sheridan, S. Robinson, D. Drukteinis, J.L. Ferrara,
and P.M. Mauch. 2001. Immunogenicity of Ly5 (CD45)-antigens ham-
pers long-term engraftment following minimal conditioning in a mu-
rine bone marrow transplantation model. Stem Cells. 19:80–87.
19. Goldman, J.P., M.P. Blundell, L. Lopes, C. Kinnon, J.P. Di Santo, and
A.J. Thrasher. 1998. Enhanced human cell engraftment in mice defi -
cient in RAG2 and the common cytokine receptor gamma chain. Br. J.
20. Akashi, K., D. Traver, T. Miyamoto, and I.L. Weissman. 2000. A clo-
nogenic common myeloid progenitor that gives rise to all myeloid line-
ages. Nature. 404:193–197.
21. Kondo, M., I.L. Weissman, and K. Akashi. 1997. Identifi cation of clo-
nogenic common lymphoid progenitors in mouse bone marrow. Cell.
84 ENGRAFTMENT IN RARE EMPTY HEMATOPOIETIC STEM CELL NICHES | Bhattacharya et al.
22. Sitnicka, E., D. Bryder, K. Theilgaard-Monch, N. Buza-Vidas, J.
Adolfsson, and S.E. Jacobsen. 2002. Key role of fl t3 ligand in regulation
of the common lymphoid progenitor but not in maintenance of the
hematopoietic stem cell pool. Immunity. 17:463–472.
23. Karsunky, H., M. Merad, A. Cozzio, I.L. Weissman, and M.G. Manz.
2003. Flt3 ligand regulates dendritic cell development from Flt3+ lym-
phoid and myeloid-committed progenitors to Flt3+ dendritic cells in
vivo. J. Exp. Med. 198:305–313.
24. Hardy, R.R., C.E. Carmack, S.A. Shinton, J.D. Kemp, and K.
Hayakawa. 1991. Resolution and characterization of pro–B and pre-
pro–B cell stages in normal mouse bone marrow. J. Exp. Med. 173:
25. Li, Y.S., R. Wasserman, K. Hayakawa, and R.R. Hardy. 1996.
Identifi cation of the earliest B lineage stage in mouse bone marrow.
26. Kondo, M., and I.L. Weissman. 2000. Function of cytokines in lym-
phocyte development. Curr. Top. Microbiol. Immunol. 251:59–65.
27. Tesch, H., F.I. Smith, W.J. Muller-Hermes, and K. Rajewsky. 1984.
Heterogeneous and monoclonal helper T cells induce similar anti-(4-
hydroxy-3-nitrophenyl)acetyl (NP) antibody populations in the primary
adoptive response. I. Isotype distribution. Eur. J. Immunol. 14:188–194.
28. Smith, F.I., H. Tesch, and K. Rajewsky. 1984. Heterogeneous and
monoclonal helper T cells induce similar anti-(4-hydroxy-3-
nitrophenyl)acetyl (NP) antibody populations in the primary adoptive
response. II. Lambda light chain dominance and idiotope expression.
Eur. J. Immunol. 14:195–200.
29. Rossi, D.J., D. Bryder, J.M. Zahn, H. Ahlenius, R. Sonu, A.J. Wagers,
and I.L. Weissman. 2005. Cell intrinsic alterations underlie hematopoi-
etic stem cell aging. Proc. Natl. Acad. Sci. USA. 102:9194–9199.
30. Shizuru, J.A., L. Jerabek, C.T. Edwards, and I.L. Weissman. 1996.
Transplantation of purifi ed hematopoietic stem cells: requirements for
overcoming the barriers of allogeneic engraftment. Biol. Blood Marrow
31. Prockop, S.E., and H.T. Petrie. 2004. Regulation of thymus size
by competition for stromal niches among early T cell progenitors.
J. Immunol. 173:1604–1611.
32. Grusby, M.J., R.S. Johnson, V.E. Papaioannou, and L.H. Glimcher.
1991. Depletion of CD4+ T cells in major histocompatibility complex
class II-defi cient mice. Science. 253:1417–1420.
33. Zijlstra, M., M. Bix, N.E. Simister, J.M. Loring, D.H. Raulet, and
R. Jaenisch. 1990. Beta 2-microglobulin defi cient mice lack CD4-8+
cytolytic T cells. Nature. 344:742–746.
34. Lamouse-Smith, E., V.K. Clements, and S. Ostrand-Rosenberg. 1993.
Beta 2M−/− knockout mice contain low levels of CD8+ cytotoxic
T lymphocyte that mediate specifi c tumor rejection. J. Immunol. 151:
35. Apasov, S., and M. Sitkovsky. 1993. Highly lytic CD8+, alpha beta
T-cell receptor cytotoxic T cells with major histocompatibility complex
(MHC) class I antigen-directed cytotoxicity in beta 2-microglobulin,
MHC class I-defi cient mice. Proc. Natl. Acad. Sci. USA. 90:
36. Bix, M., N.S. Liao, M. Zijlstra, J. Loring, R. Jaenisch, and D. Raulet.
1991. Rejection of class I MHC-defi cient haemopoietic cells by irradi-
ated MHC-matched mice. Nature. 349:329–331.
37. Antoine, C., S. Muller, A. Cant, M. Cavazzana-Calvo, P. Veys, J.
Vossen, A. Fasth, C. Heilmann, N. Wulff raat, R. Seger, et al. 2003.
Long-term survival and transplantation of haemopoietic stem cells for
immunodefi ciencies: report of the European experience 1968–99.
38. Taniguchi, H., M. Abe, T. Shirai, K. Fukao, and H. Nakauchi. 1995.
Reconstitution ratio is critical for alloreactive T cell deletion and skin
graft survival in mixed bone marrow chimeras. J. Immunol. 155:
39. Kay, M.M., and T. Makinodan. 1981. Relationship between aging and
immune system. Prog. Allergy. 29:134–181.
40. Kamminga, L.M., R. van Os, A. Ausema, E.J. Noach, E. Weersing, B.
Dontje, E. Vellenga, and G. de Haan. 2005. Impaired hematopoietic
stem cell functioning after serial transplantation and during normal ag-
ing. Stem Cells. 23:82–92.
41. Gatti, R.A., H.J. Meuwissen, H.D. Allen, R. Hong, and R.A. Good.
1968. Immunological reconstitution of sex-linked lymphopenic immu-
nological defi ciency. Lancet. 2:1366–1369.
42. Bach, F.H., R.J. Albertini, P. Joo, J.L. Anderson, and M.M. Bortin.
1968. Bone-marrow transplantation in a patient with the Wiskott-
Aldrich syndrome. Lancet. 2:1364–1366.
43. Buckner, C.D., R.B. Epstein, R.H. Rudolph, R.A. Clift, R. Storb, and
E.D. Thomas. 1970. Allogeneic marrow engraftment following whole
body irradiation in a patient with leukemia. Blood. 35:741–750.
44. Appelbaum, F.R., G.P. Herzig, J.L. Ziegler, R.G. Graw, A.S. Levine,
and A.B. Deisseroth. 1978. Successful engraftment of cryopreserved au-
tologous bone marrow in patients with malignant lymphoma. Blood. 52:
45. Thomas, E.D., H.L. Lochte Jr., W.C. Lu, and J.W. Ferrebee. 1957.
Intravenous infusion of bone marrow in patients receiving radiation and
chemotherapy. N. Engl. J. Med. 257:491–496.
46. Sayegh, M.H., N.A. Fine, J.L. Smith, H.G. Rennke, E.L. Milford,
and N.L. Tilney. 1991. Immunologic tolerance to renal allografts after
bone marrow transplants from the same donors. Ann. Intern. Med. 114:
47. Nelson, J.L., R. Torrez, F.M. Louie, O.S. Choe, R. Storb, and K.M.
Sullivan. 1997. Pre-existing autoimmune disease in patients with long-
term survival after allogeneic bone marrow transplantation. J. Rheumatol.
48. Boggs, D.R. 1984. The total marrow mass of the mouse: a simplifi ed
method of measurement. Am. J. Hematol. 16:277–286.
49. Peschon, J.J., P.J. Morrissey, K.H. Grabstein, F.J. Ramsdell, E.
Maraskovsky, B.C. Gliniak, L.S. Park, S.F. Ziegler, D.E. Williams, C.
B. Ware, et al. 1994. Early lymphocyte expansion is severely impaired
in interleukin 7 receptor–defi cient mice. J. Exp. Med. 180:1955–1960.
50. Rodewald, H.R., K. Kretzschmar, W. Swat, and S. Takeda. 1995.
Intrathymically expressed c-kit ligand (stem cell factor) is a major factor
driving expansion of very immature thymocytes in vivo. Immunity. 3:
51. Sitnicka, E., C. Brakebusch, I.L. Martensson, M. Svensson, W.W.
Agace, M. Sigvardsson, N. Buza-Vidas, D. Bryder, C.M. Cilio, H.
Ahlenius, et al. 2003. Complementary signaling through fl t3 and inter-
leukin-7 receptor α is indispensable for fetal and adult B cell genesis.
J. Exp. Med. 198:1495–1506.
52. Ranheim, E.A., H.C. Kwan, T. Reya, Y.K. Wang, I.L. Weissman, and
U. Francke. 2005. Frizzled 9 knock-out mice have abnormal B-cell de-
velopment. Blood. 105:2487–2494.
53. Balciunaite, G., R. Ceredig, H.J. Fehling, J.C. Zuniga-Pfl ucker, and A.G.
Rolink. 2005. The role of Notch and IL-7 signaling in early thymocyte
proliferation and diff erentiation. Eur. J. Immunol. 35:1292–1300.
54. Cao, X., E.W. Shores, J. Hu-Li, M.R. Anver, B.L. Kelsall, S.M. Russell,
J. Drago, M. Noguchi, A. Grinberg, E.T. Bloom, et al. 1995. Defective
lymphoid development in mice lacking expression of the common cy-
tokine receptor gamma chain. Immunity. 2:223–238.
55. DiSanto, J.P., D. Guy-Grand, A. Fisher, and A. Tarakhovsky. 1996.
Critical role for the common cytokine receptor γ chain in intrathymic
and peripheral T cell selection. J. Exp. Med. 183:1111–1118.
56. Kume, A., M. Koremoto, H. Mizukami, T. Okada, Y. Hanazono, K.
Sugamura, and K. Ozawa. 2002. Selective growth advantage of wild-
type lymphocytes in X-linked SCID recipients. Bone Marrow Transplant.
57. Shinkai, Y., G. Rathbun, K.P. Lam, E.M. Oltz, V. Stewart, M.
Mendelsohn, J. Charron, M. Datta, F. Young, A.M. Stall, et al. 1992.
RAG-2-defi cient mice lack mature lymphocytes owing to inability to
initiate V(D)J rearrangement. Cell. 68:855–867.
58. Spitzer, T.R., S.L. McAfee, B.R. Dey, C. Colby, J. Hope, H. Grossberg,
F. Preff er, J. Shaff er, S.I. Alexander, D.H. Sachs, and M. Sykes. 2003.
Nonmyeloablative haploidentical stem-cell transplantation using anti-
CD2 monoclonal antibody (MEDI-507)-based conditioning for refrac-
tory hematologic malignancies. Transplantation. 75:1748–1751.
59. Ferry, C., and G. Socie. 2003. Busulfan-cyclophosphamide versus total
body irradiation-cyclophosphamide as preparative regimen before allo-
geneic hematopoietic stem cell transplantation for acute myeloid leuke-
mia: what have we learned? Exp. Hematol. 31:1182–1186.
JEM VOL. 203, January 23, 2006
60. Buckley, R.H., S.E. Schiff , R.I. Schiff , L. Markert, L.W. Williams, J.L.
Roberts, L.A. Myers, and F.E. Ward. 1999. Hematopoietic stem-cell
transplantation for the treatment of severe combined immunodefi -
ciency. N. Engl. J. Med. 340:508–516.
61. Muller, S.M., T. Kohn, A.S. Schulz, K.M. Debatin, and W. Friedrich.
2000. Similar pattern of thymic-dependent T-cell reconstitution in in-
fants with severe combined immunodefi ciency after human leukocyte
antigen (HLA)-identical and HLA-nonidentical stem cell transplanta-
tion. Blood. 96:4344–4349.
62. Fischer, A., F. Le Deist, S. Hacein-Bey-Abina, I. Andre-Schmutz, S.
Basile Gde, J.P. de Villartay, and M. Cavazzana-Calvo. 2005. Severe
combined immunodefi ciency. A model disease for molecular immunol-
ogy and therapy. Immunol. Rev. 203:98–109.
63. Cavazzana-Calvo, M., C. Lagresle, S. Hacein-Bey-Abina, and A.
Fischer. 2005. Gene therapy for severe combined immunodefi ciency.
Annu. Rev. Med. 56:585–602.
64. McCredie, K.B., E.M. Hersh, and E.J. Freireich. 1971. Cells capable of
colony formation in the peripheral blood of man. Science. 171:293–294.
65. Goodman, J.W., and G.S. Hodgson. 1962. Evidence for stem cells in
the peripheral blood of mice. Blood. 19:702–714.
66. Storek, J., R.P. Witherspoon, D. Webb, and R. Storb. 1996. Lack of
B cells precursors in marrow transplant recipients with chronic graft-
versus-host disease. Am. J. Hematol. 52:82–89.
67. Wall, D.A., S.D. Hamberg, D.S. Reynolds, S.J. Burakoff , A.K. Abbas,
and J.L. Ferrara. 1988. Immunodefi ciency in graft-versus-host disease. I.
Mechanism of immune suppression. J. Immunol. 140:2970–2976.