Hematopoietic stem cells do not depend on N-cadherin to regulate their maintenance

doi:10.1016/j.stem.2008.10.005

Cell Stem Cell. Author manuscript; available in PMC 2009 August 6.

Published in final edited form as:

Cell Stem Cell. 2009 February 6; 4(2): 170–179.

doi: 10.1016/j.stem.2008.10.005

PMCID: PMC2681089

HHMIMSID: HHMIMS99882

Hematopoietic stem cells do not depend on N-cadherin to regulate their maintenance

Mark J. Kiel,¹ Melih Acar,¹ Glenn L. Radice,² and Sean J. Morrison^1,³

¹ Howard Hughes Medical Institute, Department of Internal Medicine, and Center for Stem Cell Biology, Life Sciences Institute, University of Michigan, Ann Arbor, Michigan, 48109-2216

² Center for Translational Medicine, Department of Medicine, Jefferson Medical College, Philadelphia, PA 19107

³ Contact: 5435 Life Sciences Institute, 210 Washtenaw Ave., Ann Arbor, Michigan, 48109-2216; phone 734-647-6261; fax 734-615-8133; email seanjm/at/umich.edu

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SUMMARY

According to the “osteoblastic niche” model, hematopoietic stem cells (HSCs) are maintained by N-cadherin-mediated homophilic adhesion to osteoblasts at the bone marrow endosteum. In contrast to this model, we cannot detect N-cadherin expression by HSCs and most HSCs do not localize to the endosteal surface. It has nonetheless been suggested that HSCs express low levels of N-cadherin that regulate HSC maintenance. To test this we conditionally deleted N-cadherin from HSCs and other hematopoietic cells in adult Mx-1-Cre⁺N-cadherin^fl/⁻ mice. N-cadherin deficiency had no detectable effect on HSC maintenance or hematopoiesis. N-cadherin deficiency did not affect bone marrow cellularity or lineage composition, the numbers of colony-forming progenitors, the frequency of HSCs, the ability of HSCs to sustain hematopoiesis over time, or their ability to reconstitute irradiated mice in primary or secondary transplants. Loss of N-cadherin does not lead to HSC depletion. N-cadherin expression by HSCs is not necessary for niche function.

INTRODUCTION

HSCs persist throughout adult life in the bone marrow where they continuously produce new blood cells to maintain hematopoiesis. To understand the mechanisms that sustain HSCs it is necessary to identify the niche – the specialized microenvironment in which HSCs are thought to reside (Adams and Scadden, 2006; Kiel and Morrison, 2008).

Osteoblasts are thought to contribute to HSC niches. Genetic manipulations that increase osteoblast numbers in mice also increase the number of HSCs (Calvi et al., 2003; Zhang et al., 2003). Osteoblasts have also been proposed to secrete factors that are necessary for HSC maintenance, including angiopoietin, thrombopoietin, and CXCL12 (Arai et al., 2004; Yoshihara et al., 2007), though none of these factors have yet been conditionally deleted from osteoblasts and each is also secreted by other cell types. Calcium ions from bone (due to osteoblast and osteoclast activity) also regulate HSC localization and maintenance (Adams et al., 2006). These observations raise two general possibilities for how osteoblasts could contribute to HSC maintenance. One possibility is that osteoblasts produce extracellular factors that diffuse into the marrow, directly or indirectly regulating HSC niches that are near, but not at, the endosteum. A second possibility is that osteoblasts directly promote HSC maintenance by binding HSCs, creating “osteoblastic niches” at the endosteal surface.

The possibility of bone marrow HSC niches near, but not at, the endosteum has been raised by a number of recent observations (Kiel and Morrison, 2008). We have localized HSCs within the bone marrow using SLAM family markers that give high levels of stem cell purity and found that few HSCs localize to the endosteal surface itself (Kiel et al., 2005; Kiel et al., 2007b). Instead, most HSCs are present around sinusoids, some of which are close to the endosteum while others are more distant. It remains uncertain whether perivascular cells directly promote HSC maintenance; however, the observation that HSCs are more likely than other hematopoietic cells to be adjacent to sinusoids (Kiel et al., 2007b) raises the possibility that there are perivascular niches. Consistent with this possibility, recent studies have suggested that perivascular cells, such as reticular cells and mesenchymal progenitors, express more CXCL12 and angiopoietin than osteoblasts (Sugiyama et al., 2006; Sacchetti et al., 2007). This raises the question of whether HSCs are maintained in direct contact with osteoblasts, or whether they are maintained in other microenvironments that are directly or indirectly influenced by factors secreted by endosteal cells.

The widely discussed osteoblastic niche model has favored the idea that HSCs are maintained in direct contact with osteoblasts (Zhang et al., 2003; Suda et al., 2005; Wilson and Trumpp, 2006; Zhang and Li, 2008). A fundamental element of this model is that HSCs adhere to the surface of osteoblasts via N-cadherin mediated homophilic adhesion and that osteoblasts directly promote the maintenance of HSCs by mechanisms that involve cell-cell contact, including Notch and N-cadherin activation (Zhang et al., 2003; Wilson et al., 2004; Haug et al., 2008). However, genetic evidence supporting such mechanisms is lacking. Conditional deletion of Jagged1 and/or Notch1 (the ligand/receptor combination proposed to regulate Notch signaling between osteoblasts and HSCs) does not affect HSC maintenance or function (Mancini et al., 2005). Conditional inactivation of all canonical Notch signaling by disruption of the CSL/Rbp-J transcriptional complex also does not affect the maintenance or function of adult HSCs (Maillard et al., 2008). It remains possible that Notch could regulate HSCs through some non-canonical signaling pathway, though we are not aware of any data that yet support this possibility. It has not been tested whether N-cadherin deficiency affects HSC maintenance.

Given the central role proposed for N-cadherin in the creation of osteoblastic niches, we recently tested whether HSCs express N-cadherin. We were unable to detect N-cadherin expression among highly purified HSCs by quantitative PCR, by staining with commercially available anti-N-cadherin antibodies, or in N-cadherin:LacZ genetrap mice (Kiel et al., 2007b). Published microarray analyses of HSCs from multiple laboratories also failed to detect N-cadherin expression (Ivanova et al., 2002; Kiel et al., 2005). Only bone marrow cells negative for N-cadherin staining had the capacity to give long-term multilineage reconstitution of irradiated mice (Kiel et al., 2007b), even when we used the commercially available anti-N-cadherin antibody that had been used to identify osteoblastic niches in bone marrow sections (Zhang et al., 2003; Wilson et al., 2004). This suggested that HSCs could not adhere to osteoblasts via N-cadherin and that the N-cadherin⁺ cells imaged at the endosteum in prior studies could not have been HSCs. However, it was subsequently suggested that N-cadherin is expressed at low levels on HSCs and that it remains functionally important for their localization within a quiescent osteoblastic niche (Haug et al., 2008; Zhang and Li, 2008).

To resolve this controversy it was suggested that N-cadherin should be genetically deleted from adult hematopoietic cells (Hooper et al., 2007). If N-cadherin is not expressed by HSCs, then N-cadherin deficiency should not affect HSC maintenance. In contrast, if HSC niches are regulated by low levels of N-cadherin expression within HSCs then N-cadherin deletion should lead to the depletion of HSCs and to deficits in hematopoiesis. We have now addressed this by conditionally deleting N-cadherin from HSCs and other hematopoietic cells in adult Mx-1-Cre⁺N-cadherin^fl/⁻ mice. Despite efficiently deleting N-cadherin from HSCs, these mice exhibited no detectable effects of N-cadherin deficiency on HSC maintenance or hematopoiesis, either acutely after N-cadherin deletion or several months later. Bone marrow cells from these mice exhibited normal HSC frequency and a normal capacity to reconstitute irradiated mice, even upon transplantation into secondary recipients. N-cadherin is therefore not required cell-autonomously to regulate HSC maintenance or function. It remains possible that osteoblasts regulate HSC maintenance through other mechanisms, but HSC maintenance does not depend upon N-cadherin-mediated homophylic adhesion to osteoblasts, as proposed in current osteoblastic niche models.

RESULTS

N-cadherin is efficiently deleted after pIpC treatment

Mice with germline N-cadherin deficiency (N-cadherin^−/−) die by E11 due to severe developmental defects including cardiovascular failure (Radice et al., 1997). Therefore, to study the effects of N-cadherin deficiency on adult HSCs, we mated previously described N-cadherin^fl mice (Kostetskii et al., 2005; Luo et al., 2006; Kadowaki et al., 2007; Li et al., 2008) with Mx-1-Cre mice (Kuhn et al., 1995) to conditionally delete N-cadherin from adult HSCs. Each of these strains was backcrossed for at least 6 generations onto a C57BL/6 background. Mx-1-Cre expression is activated in HSCs, other hematopoietic cells, and some other tissues by administration of polyinosine-polycytidine (pIpC) (Kuhn et al., 1995; Hock et al., 2004; Yilmaz et al., 2006b).

To test the efficiency of N-cadherin excision within HSCs, we cultured individual CD150⁺CD48⁻CD41⁻ lineage⁻Sca-1⁺c-kit⁺ HSCs (1 cell/well) isolated from Mx-1-Cre⁺N-cadherin^fl/⁻ mice after they had been treated with 7 doses of pIpC over 14 days. These cells include more than 95% of all long-term multilineage reconstituting cells in the bone marrow, and more than 40% of single cells within this population give long-term multilineage reconstitution after transplantation into irradiated mice (Kiel et al., 2005; Yilmaz et al., 2006a; Kiel et al., 2008). Genomic DNA was extracted from individual myeloerythroid colonies formed by these HSCs and analyzed by PCR. Of colonies cultured immediately after pIpC treatment, 53 of 54 showed complete deletion of N-cadherin^fl (Fig. 1A,B). Of colonies that arose from HSCs cultured 1 to 3 months following pIpC treatment, 72 of 73 showed complete deletion of the N-cadherin^fl allele (Fig. 1A,B). This indicates that at least 98% of HSCs deleted N-cadherin upon pIpC treatment. The rare non-recombined cells did not appear to have a competitive advantage over N-cadherin deficient HSCs, as their frequency did not increase with time after pIpC treatment.

Figure 1

pIpC treatment of adult Mx-1-Cre⁺N-cadherin^fl/⁻ mice led to efficient N-cadherin deletion from HSCs without altering hematopoiesis

To ensure the accurate detection of N-cadherin excision we sequenced the PCR products corresponding to the wild-type, floxed and deleted N-cadherin alleles and confirmed 100% identity to the appropriate regions of the N-cadherin gene (data not shown). Moreover, we designed independent PCR primers that specifically amplified a region of N-cadherin (the first exon) that is deleted upon Cre-mediated excision and confirmed excision of the floxed region of N-cadherin in hematopoietic colonies from pIpC treated Mx-1-Cre⁺N-cadherin^fl/⁻ mice (Suppl. Fig. 1). pIpC treatment of Mx-1-Cre⁺N-cadherin^fl/⁻ mice therefore leads to N-cadherin deletion.

N-cadherin deficiency does not alter hematopoiesis

If N-cadherin is critical for HSC maintenance then N-cadherin deficiency should lead to defects in hematopoiesis. We therefore tested whether acute or chronic loss of N-cadherin affected hematopoiesis in Mx-1-Cre⁺N-cadherin^fl/⁻ mice. To test this we harvested peripheral blood and bone marrow from Mx-1-Cre⁺N-cadherin^fl/⁻ mice or littermate controls (mice lacking Cre or bearing at least one wild-type allele of N-cadherin) either immediately following pIpC treatment (2 weeks pIpC; Figure 1C–G) or 1–3 months following 2 weeks of pIpC administration (Figure 1H-L). We did not detect any effect of N-cadherin deletion on white blood cell (WBC), erythrocyte (RBC) or platelet (Plts) concentration in the blood of Mx-1-Cre⁺N-cadherin^fl/⁻ mice as compared to littermate controls at either time point (Fig. 1C, H). We also did not detect any effect of N-cadherin deficiency on bone marrow cellularity (Fig. 1D, I) or composition with respect to the erythroid (Ter119⁺ cells), myeloid (Mac-1⁺Gr-1⁺), B (B220⁺surfaceIgM^±) or T cell (CD3⁺) lineages (Fig. 1E, J). Finally, we did not detect any effect of N-cadherin deficiency on the frequency (Fig. 1F, K) or types (Fig. 1G, L) of colony-forming progenitors in the bone marrow. These results indicate that N-cadherin is not required within HSCs for hematopoiesis.

N-cadherin deficiency does not alter HSC frequency or function in vivo

To directly test whether N-cadherin deficiency affects HSC maintenance we examined the HSC content of bone marrow isolated from Mx-1-Cre⁺N-cadherin^fl/⁻ mice or littermate controls either immediately following pIpC treatment (Fig. 2A–C) or 1–3 months later (Fig. 2D–F). As previously reported for wild-type mice (Kiel et al., 2005; Kiel et al., 2007a; Kiel et al., 2008), the vast majority of CD150⁺CD48⁻CD41⁻ lineage⁻ cells from the bone marrow of Mx-1-Cre⁺N-cadherin^fl/⁻ mice or littermate controls were positive for the HSC markers Sca-1 and c-kit (Fig. 2A, D). We did not detect any difference in the frequency (Fig. 2B) or absolute number (Fig. 2E) of CD150⁺CD48⁻CD41⁻ lineage⁻Sca-1⁺c-kit⁺ HSCs in the hindlimb bone marrow of Mx-1-Cre⁺N-cadherin^fl/⁻ mice as compared to littermate controls. We were also unable to detect any difference in the frequency or absolute number of Flk2⁻ lineage⁻Sca-1⁺c-kit⁺ HSCs in Mx-1-Cre⁺N-cadherin^fl/⁻ mice as compared to littermate controls (Fig. 3). Finally, when CD150⁺CD48⁻CD41⁻ lineage⁻Sca-1⁺c-kit⁺ HSCs were individually cultured in methylcellulose, 83–89% of cells formed large myeloerythroid colonies irrespective of whether they were from Mx-1-Cre⁺N-cadherin^fl/⁻ mice or littermate controls (Fig. 2C, F). We therefore could not detect any effect of N-cadherin deficiency on the frequency, absolute number, or colony-forming capacity of HSCs either immediately after N-cadherin deletion or 1–3 months later.

Figure 2

N-cadherin deletion did not affect HSC frequency or the ability of HSCs to form colonies in culture

Figure 3

N-cadherin deficiency did not affect the frequency of Flk2⁻ lineage⁻Sca-1⁺c-kit⁺ HSCs in the bone marrow

In case N-cadherin protein turns over slowly or the consequences of its absence only become apparent over long periods of time we treated some mice with pIpC for 2 weeks then waited for 5 months before analyzing Mx-1-Cre⁺N-cadherin^fl/⁻ mice or littermate controls (Fig. 4). Five months after N-cadherin deletion, we observed no effect of N-cadherin deficiency on WBC, RBC, or platelet levels in the blood (Fig. 4A) or on bone marrow cellularity (Fig. 4B) or composition (Fig. 4C). We also did not detect any effect of N-cadherin deficiency on the frequency or absolute number of CD150⁺CD48⁻CD41⁻ lineage⁻Sca-1⁺c-kit⁺ HSCs (Fig. 4D,E) or Flk2⁻ lineage⁻Sca-1⁺c-kit⁺ HSCs (Fig. 4F,G). These data indicate that even prolonged absence of N-cadherin does not affect hematopoiesis or HSC maintenance.

Figure 4

Prolonged N-cadherin deficiency did not affect HSC frequency or hematopoiesis

To test whether N-cadherin deficiency affects hematopoietic recovery after myelosuppression we administered 5-fluorouracil (5-FU) to Mx-1-Cre⁺N-cadherin^fl/⁻ mice and littermate controls following 2 weeks of pIpC administration and analyzed hematopoiesis 10 days later. We observed no differences in peripheral blood cell counts or in the cellularity or composition of the bone marrow or spleen of Mx-1-Cre⁺N-cadherin^fl/⁻ mice as compared to littermate controls (Suppl. Fig. 2A–C). We also observed no difference in the frequency or absolute number of CD150⁺CD48⁻CD41⁻ lineage⁻Sca-1⁺c-kit⁺ cells in the bone marrow or spleens of Mx-1-Cre⁺N-cadherin^fl/⁻ mice as compared to control littermates (Suppl. Fig. 2D–H). We therefore could not detect any effect of N-cadherin deficiency on hematopoiesis following myelosuppression.

N-cadherin deficiency does not affect HSC maintenance over time in vivo

To test whether N-cadherin deficiency affects HSC function in vivo, we competitively transplanted 1×10⁶ bone marrow cells from non-pIpC-treated CD45.2⁺ Mx-1-Cre⁺N-cadherin^fl/⁻ mice or CD45.2⁺ littermate controls into lethally irradiated CD45.1⁺ recipients along with 3×10⁵ wild-type CD45.1⁺ bone marrow cells. Four weeks following transplantation, we analyzed the blood of these recipients for engraftment by CD45.2⁺ donor cells and found that both Mx-1-Cre⁺N-cadherin^fl/⁻ cells and control cells had engrafted at similar levels, as expected (Fig. 5A, C, E; 4 week time point). Starting 6 weeks after transplantation, we administered pIpC for 2 weeks to all mice to delete N-cadherin. If N-cadherin is required for HSC maintenance or function in vivo then we would expect to observe a loss of donor cells over time in the mice transplanted with Mx-1-Cre⁺N-cadherin^fl/⁻ cells as these cells should exhibit a competitive disadvantage relative to wild-type HSCs that engrafted in the same mice. We monitored the levels of donor cell reconstitution in all recipients for a total of 20 weeks after pIpC treatment but never observed a decline in donor myeloid (Mac-1⁺; Fig. 5A, B), B (B220⁺; Fig. 5C, D) or T cells (CD3⁺; Fig. 5E, F) in recipients of Mx-1-Cre⁺N-cadherin^fl/⁻ cells. To ensure that N-cadherin had been efficiently deleted in this experiment, we performed PCR on genomic DNA from donor-derived Mac-1⁺ myeloid cells isolated from the peripheral blood of these recipients. We observed complete excision of N-cadherin from the myeloid cells collected from recipients of Mx-1-Cre⁺N-cadherin^fl/⁻ cells but not recipients of control cells (Fig. 5G). These results indicate that N-cadherin is not required for the maintenance of HSC function in vivo and that N-cadherin-deficient HSCs are not at a competitive disadvantage relative to wild-type cells.

Figure 5

N-cadherin deficiency did not affect HSC maintenance or function in vivo

N-cadherin is not required for HSC reconstitution of irradiated recipients

If N-cadherin is required for HSC homing or engraftment in the niche, then deletion of N-cadherin from HSCs prior to transplantation should cause an engraftment defect. To test this we treated CD45.2⁺ Mx-1-Cre⁺N-cadherin^fl/⁻ mice or littermate controls with pIpC, then transplanted 1×10⁶ bone marrow cells 3 months later along with 3×10⁵ recipient bone marrow cells into CD45.1⁺ recipient mice. All of the recipients of both Mx-1-Cre⁺N-cadherin^fl/⁻ cells (n=12) and control cells (n=10) became long-term multilineage reconstituted by donor myeloid, B, and T cells (Fig. 6A). The level of reconstitution by donor cells did not differ between recipients of Mx-1-Cre⁺N-cadherin^fl/⁻ cells and control cells in terms of all donor blood cells (CD45.2⁺, Fig. 6B, p=0.16), donor myeloid cells (Mac-1⁺, Fig. 6C, D, p=0.18), B cells (B220⁺, Fig. 6E, F, p=0.19), or T cells (CD3⁺, Fig. 6G, H, p=0.16). N-cadherin is therefore not required for the homing or engraftment of HSCs in vivo.

Figure 6

N-cadherin deficiency did not affect the ability of HSCs to engraft or to reconstitute irradiated recipients

To even more stringently test whether N-cadherin deficiency affects HSC function in vivo we also performed secondary transplants from the primary recipients described in Figure 5. Twenty-eight weeks after transplantation into the primary recipients, we transplanted 1×10⁶ bone marrow cells from primary recipients of CD45.2⁺ Mx-1-Cre⁺N-cadherin^fl/⁻ mice or CD45.2⁺ littermate controls into lethally irradiated CD45.1⁺ secondary recipients along with 3×10⁵ wild-type CD45.1⁺ bone marrow cells. We monitored the blood of the secondary recipients for engraftment by CD45.2⁺ donor myeloid (Mac-1⁺; Fig. 7A, B), B (B220⁺; Fig. 7C, D) and T cells (CD3⁺; Fig. 7E, F) for a total of 17 weeks after transplantation. We found no difference in the level of donor cell engraftment between mice that received Mx-1-Cre⁺N-cadherin^fl/⁻ donor cells as compared to control cells in any lineage. To ensure that mice were reconstituted by N-cadherin deficient donor cells we performed PCR on genomic DNA from CD45.2⁺ myeloid cells isolated from the peripheral blood of these recipients. As expected, we observed complete excision of N-cadherin from the myeloid cells collected from recipients of Mx-1-Cre⁺N-cadherin^fl/⁻ cells (Fig. 7G).

Figure 7

N-cadherin deficiency did not affect HSC function upon serial transplantation into secondary recipients

We also performed another secondary transplant experiment using the mice shown in Figure 6 (mice in which N-cadherin was deleted prior to transplantation into the primary recipients). Four primary recipients of N-cadherin deficient cells (averaging 74±13% of white blood cells that were donor derived) and four primary recipients of control cells (averaging 75±8% of white blood cells that were donor derived) were selected for secondary transplantation. Twenty-eight weeks after transplantation into the primary recipients, 3×10⁶ bone marrow cells from each primary recipient were transplanted into irradiated secondary recipients (5–6 secondary recipients per primary recipient). Six weeks later, all of the secondary recipients were found to have high levels of multilineage reconstitution by donor cells, irrespective of the genotype of the donor cells. Secondary recipients of N-cadherin deficient cells averaged 73±20% of white blood cells that were donor-derived while secondary recipients of control cells averaged 65±29% of white blood cells that were donor-derived. Together with the results in Figure 7, these results indicate that N-cadherin is not required for the maintenance of HSCs in vivo and that N-cadherin deficiency does not affect the ability of HSCs to reconstitute primary or secondary recipient mice.

DISCUSSION

The “osteoblastic niche” model proposes that HSCs are maintained in direct contact with osteoblasts via N-cadherin mediated homophilic adhesion (Zhang et al., 2003; Arai et al., 2004; Wilson et al., 2004; Suda et al., 2005; Wilson and Trumpp, 2006; Haug et al., 2008; Zhang and Li, 2008). According to this model, N-cadherin is postulated to be required within HSCs to maintain adhesion with the niche, to regulate quiescence, to regulate β-catenin signaling, and to maintain HSCs in an undifferentiated state (Zhang et al., 2003; Arai et al., 2004; Wilson et al., 2004; Suda et al., 2005; Wilson and Trumpp, 2006; Haug et al., 2008; Zhang and Li, 2008). However, these predictions have not been tested in N-cadherin deficient mice. Our data demonstrate that N-cadherin deletion in vivo from HSCs and other hematopoietic cells has no effect on hematopoiesis in the bone marrow (Figs. 1, ,4),4), HSC frequency (Figs. 2, ,3,3, ,4),4), HSC maintenance or function over time (Fig. 5), or on the ability of HSCs to engraft and reconstitute irradiated mice in primary or secondary transplants (Fig. 6, ,7).7). In no assay did N-cadherin deficient HSCs show a competitive disadvantage relative to wild-type HSCs. N-cadherin is therefore not required autonomously within HSCs to regulate their maintenance or function.

Other data have also been inconsistent with the idea that HSCs are maintained as a result of cell-cell contact with osteoblasts. HSCs were originally localized to the surface of osteoblasts based on staining for N-cadherin⁺ bromo-deoxyuridine (BrdU) label-retaining cells (Zhang et al., 2003). However, in addition to being unable to detect N-cadherin expression within HSCs by several different techniques (Kiel et al., 2007b), we have also found that BrdU label-retention has very poor specificity and very poor sensitivity as an HSC marker: the vast majority of HSCs fail to retain BrdU for long periods of time and the vast majority of bone marrow cells that do retain BrdU are not HSCs (Kiel et al., 2007a). When we localize highly purified HSCs within bone marrow sections using SLAM family markers, we find a minority of HSCs that localize near the endosteal surface (<20%) and a majority of HSCs (>60%) that localize to sinusoids that are not at the endosteal surface (Kiel et al., 2005; Kiel et al., 2007b). It thus remains possible that a subset of HSCs is maintained in niches that are at, or near, the endosteum by N-cadherin-independent mechanisms but it seems unlikely that all HSCs depend on contact with osteoblasts for their maintenance. Rather, our data (Kiel et al., 2005; Kiel et al., 2007b) and the data of others (Sugiyama et al., 2006; Sacchetti et al., 2007) raise the possibility of perivascular niches for HSCs, though direct evidence that perivascular cells promote HSC maintenance is also lacking. It is also important to note that even if a subset of HSCs reside in perivascular niches, these niches may depend on factors secreted by osteoblasts for their creation or maintenance.

A recent study from Li and colleagues (Haug et al., 2008) used an anti-N-cadherin antibody, MNCD2 (Matsunami and Takeichi, 1995), that was not used in prior studies of HSCs to suggest that HSCs do express N-cadherin at low levels and that this antibody can be used to resolve HSCs into two populations: an N-cadherin^low population that contains long-term multilineage reconstituting activity and a distinct N-cadherin^intermediate “reserve” population that resides within the osteoblastic niche. However, the N-cadherin^low staining was not clearly distinct from background and the N-cadherin^intermediate population had little HSC activity in reconstitution assays. We have independently tested the MNCD2 antibody and fail to detect staining of HSCs above background (Suppl. Fig. 3) irrespective of the age of mice used for HSC isolation (Suppl. Fig. 4) or whether the HSCs were isolated by collagenase digestion of the bones followed by staining with MNCD2 in the presence of calcium (Suppl. Fig. 5). Finally, we readily detect N-cadherin expression by 30,000 neonatal forebrain cells by Western blot, but we have been unable to detect N-cadherin expression within 100,000 whole bone marrow cells or 100,000 Lineage⁻Sca-1⁺c-kit⁺ cells in the same Western blots (Suppl. Fig. 7–9). These results are consistent with our previously reported conclusion that N-cadherin expression cannot be detected in HSCs (Kiel et al., 2007b).

The recent study from Li and colleagues (Haug et al., 2008) used MNCD2 to evaluate N-cadherin expression by HSCs using flow-cytometry, even though the specificity of MNCD2 antibody for N-cadherin has only ever been tested by Western blot (Radice et al., 1997; Haug et al., 2008). In an attempt to resolve the discrepancies between our results and their results we tested the specificity of MNCD2 antibody by flow-cytometry. We observe staining of some bone marrow cells with the MNCD2 antibody by flow-cytometry (Suppl. Fig. 3), particularly among B220⁺ B-cells that express surface IgM (sIgM; Suppl. Fig. 6). However, the MNCD2 staining of bone marrow cells by flow-cytometry was not affected by N-cadherin deletion (Suppl. Figs. 3, 6), and we did confirm that MNCD2 stained bone marrow cells had deleted N-cadherin by PCR of genomic DNA (Suppl. Fig. 6C). This suggests that MNCD2 staining by flow-cytometry does not reflect N-cadherin expression. We readily detected N-cadherin by Western blot in 30,000 neonatal forebrain cells using MNCD2 (Suppl. Fig. 7–9). However, neither 100,000 sIgM+ B cells (Suppl. Fig. 7) nor 100,000 of the bone marrow cells that stained with MNCD2 by flow-cytometry (Suppl. Fig. 9) exhibited detectable N-cadherin expression by Western blot using MNCD2. This indicates that MNCD2 staining reflects something other than N-cadherin in the bone marrow by flow-cytometry.

Our results indicate that MNCD2 antibody cannot be used to reliably detect N-cadherin by flow-cytometry. Our results by Western blot are thus consistent with the results that we have obtained by microarray analysis, qPCR, by flow-cytometry, and by analysis of N-cadherin gene trap mice in indicating that N-cadherin is not expressed by HSCs (Kiel et al., 2007b).

It remains possible that N-cadherin deficiency in osteoblasts could affect HSC maintenance or hematopoiesis, at least indirectly. Expression of dominant negative N-cadherin (that may affect other cadherins as well) in osteoblasts leads to a reduction in trabecular bone and defects in osteoblast maturation (Cheng et al., 2000). Effects on hematopoiesis have not yet been tested in these mice, but it is possible that defects in osteogenesis could lead to changes in HSC frequency or hematopoiesis. Nonetheless, many direct and indirect mechanisms could potentially account for such effects (Kiel and Morrison, 2008) so this would not provide evidence that HSCs are maintained via N-cadherin-mediated adhesion with osteoblasts.

Many potential models of the HSC niche remain compatible with existing data and there is little experimental basis on which to favor any specific model (Kiel and Morrison, 2008; Morrison and Spradling, 2008). One possibility is that HSCs reside in niches near the endosteum that are created through the combined action of factors secreted by endosteal, perivascular, and potentially other cells. Another possibility is that HSCs reside primarily in perivascular niches, at least some of which are close to the endosteum, and potentially influenced by factors secreted by endosteal cells. A third possibility is that there are spatially distinct niches in the bone marrow, some of which are close to the endosteum while others are not. These possibilities are not mutually exclusive and are not the only possibilities. Although some have recently proposed that it may be possible to distinguish between cells that reside in perivascular versus endosteal niches, there is little direct experimental support for this idea. Moreover, this model is conceptually problematic given that the endosteum is among the most highly vascularized sites in the bone marrow: HSCs cannot localize to the endosteum without being perivascular. To more clearly define the niche it will be necessary to genetically determine the physiologically important sources for factors that are required for HSC maintenance.

METHODS

Mice

C57BL/Ka-Thy-1.1 (CD45.2) and C57BL/Ka-Thy-1.2 (CD45.1) mice were housed in the Unit for Laboratory Animal Medicine at the University of Michigan. Both Mx-1-Cre mice and N-cadherin^fl/⁻ mice were backcrossed for at least six generations onto a C57BL/6 background. In every experiment, littermate control mice were gender-matched and either lacked Mx-1-Cre or had at least one wild-type allele of N-cadherin.

pIpC administration

pIpC was administered to mice as previously described (Yilmaz et al., 2006b). Briefly, pIpC was resuspended in Dulbecco’s phosphate buffered saline (D-PBS) at 2 mg/ml and mice were injected with 25ug per gram of body weight every other day for 14 days.

Flow-cytometry and the isolation of HSCs

Bone marrow cells were flushed from the long bones (tibias and femurs) with Hank’s buffered salt solution without calcium or magnesium, supplemented with 2% heat-inactivated calf serum (GIBCO, Grand Island, NY; HBSS+). Cells were triturated and filtered through nylon screen (45 um, Sefar America, Kansas City, MO) to obtain a single cell suspension.

For isolation of Flk2⁻ lineage⁻Sca-1⁺c-kit⁺ cells, whole bone marrow cells were incubated with PE-conjugated monoclonal antibodies to lineage markers including B220 (6B2), CD3 (KT31.1), CD4 (GK1.5), CD8 (53–6.7), Gr-1 (8C5), Mac-1 (M1/70), Ter119 and IgM in addition to FITC-conjugated anti-Sca-1 (Ly6A/E; E13-6.7) and PE-Cy5-conjugated or biotin-conjugated anti-c-kit (2B8). An antibody against Flk-2 (A2F10.1) was used to isolate Flk-2⁻ progenitors. Biotin-conjugated c-kit staining was visualized using streptavidin APC-Cy7.

For isolation of CD150⁺CD48⁻CD41⁻ lineage⁻Sca-1⁺c-kit⁺ HSCs, bone marrow cells were incubated with PE-conjugated anti-CD150 (TC15-12F12.2; BioLegend, San Diego, California), FITC-conjugated anti-CD48 (HM48-1; BioLegend), FITC-conjugated anti-CD41 (MWReg30; BD Pharmingen, San Diego, California), biotin-conjugated or APC-conjugated anti-Sca-1 (Ly6A/E; E13-6.7), and PE-Cy5-conjugated anti-c-kit (2B8) antibody, in addition to antibodies against the following FITC-conjugated lineage markers: Ter119, B220 (6B2), Gr-1 (8C5) and CD2 (RM2-5). Biotin-conjugated Sca-1 was visualized using streptavidin-conjugated APC-Cy7. HSCs were sometimes pre-enriched by selecting Sca-1⁺ or c-kit⁺ cells using paramagnetic microbeads and autoMACS (Miltenyi Biotec, Auburn, CA). Biotin-conjugated MNCD2 anti-N-cadherin antibody was obtained from Dr. Linheng Li and stored at −20°C. Non-viable cells were excluded from sorts and analyses using the viability dye DAPI (1ug/ml).

Long-term competitive repopulation assay

Adult recipient mice (CD45.1) were irradiated with an Orthovoltage X-ray source delivering approximately 300 rad/min in two equal doses of 570 rad, delivered at least 2 hr apart. Cells were injected into the retro-orbital venous sinus of anesthetized recipients. Each recipient mouse received 3×10⁵ CD45.1 marrow cells for radioprotection. Beginning 4 weeks after transplantation and continuing for at least 16 weeks, blood was obtained from the tail veins of recipient mice, subjected to ammonium-chloride potassium red cell lysis, and stained with directly conjugated antibodies to CD45.2 (104, FITC), B220 (6B2), Mac-1 (M1/70), CD3 (KT31.1), and Gr-1 (8C5) to monitor engraftment.

N-cadherin excision analysis

Genomic DNA from individual CD150⁺CD48⁻CD41⁻ lineage⁻Sca-1⁺c-kit⁺ HSC colonies or Mac-1⁺ peripheral blood cells was isolated by phenol-chloroform extraction. Glycogen (Roche, Indianapolis IN) was used to enhance the recovery of the DNA. PCR products were separated using 2% agarose gels to confirm the presence or absence of bands corresponding to wild-type, floxed or deleted alleles. Primer sequences for amplification of N-cadherin alleles were as described previously (Kostetskii et al., 2005): N-cadherin NC23 5′-GTA TGG CCA AGT AAT GGG GAC, N-cadherin L07 5′-TGC TGG TAG CAT TCC TAT GG and N-cadherin L08 5′-TAC AAG TTT GGG TGA CAA GC.

Methylcellulose culture

Sorted bone marrow cells and twice-sorted HSCs were plated in individual wells of 96-well plates (Corning, Corning NY) containing 100ul 1.0% methylcellulose (Stem Cell Technologies, Vancouver, BC) as previously described. The methylcellulose was supplemented with 1% penicillin/streptomycin (GIBCO), 50ng/ml stem cell factor (SCF), 10ng/ml interleukin-3 (IL-3), 10ng/ml interleukin-6 (IL-6), and 3U/ml erythropoietin (Epo). Colonies were maintained at 37°C in humidified incubators at 6% O₂. Colony formation was scored after 10–14 days of culture.

Supplementary Material

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Acknowledgments

This work was supported by the Howard Hughes Medical Institute and the U.S. Army Research Office (DAAD19-03-1-0168). MJK was supported by a fellowship from the University of Michigan (UM) Cancer Biology program. Thanks to David Adams, Martin White and the UM Flow-cytometry Core. Flow-cytometry was supported in part by the UM-Comprehensive Cancer, NIH CA46592. Thanks to Elizabeth Smith (Hybridoma Core Facility) for antibody production, supported in part through the Rheumatic Core Disease Center (P30 AR48310).

Footnotes

AUTHOR CONTRIBUTIONS

MJK performed most experiments and participated in the design and interpretation of experiments. MA performed experiments in Suppl. Figures 4C, 5C, 7, 8, and 9 as well as one of the secondary transplantation experiments. Glenn Radice provided N-cadherin^fl/⁻ mice and commented on experiments to assess the specificity of MNCD2 antibody. SJM participated in the design and interpretation of experiments and wrote the paper with MJK and MA.

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