LETTERS Detection of functional haematopoietic stem cell niche using real-time imaging Yucai Xie1,2*, Tong Yin1*, Winfried Wiegraebe1, Xi C. He1, Diana Miller3, Danny Stark1, Katherine Perko1, Richard Alexander1, Joel Schwartz1, Justin C. Grindley1, Jungeun Park1, Jeff S. Haug1, Joshua P. Wunderlich1, Hua Li1, Simon Zhang1, Teri Johnson1, Ricardo A. Feldman3 & Linheng Li1,4 Haematopoietic stem cell (HSC) niches, although proposed decades ago1, have only recently been identified as separate osteoblastic and vascular microenvironments2���6. Their interrelationships and inter- actions with HSCs in vivo remain largely unknown. Here we report the use of a newly developed ex vivo real-time imaging technol- ogy and immunoassaying to trace the homing of purified green- fluorescent-protein-expressing (GFP1) HSCs. We found that transplantedHSCstendedtohometotheendosteum(aninnerbone surface) in irradiated mice, but were randomly distributed and unstable in non-irradiated mice. Moreover, GFP1 HSCs were more frequently detected in the trabecular bone area compared with compact bone area, and this was validated by live imaging biolu- minescence driven by the stem-cell-leukaemia (Scl) promoter��� enhancer7. HSCs home to bone marrow through the vascular system. We found that the endosteum is well vascularized and that vasculature is frequently localized near N-cadherin1 pre- osteoblastic cells, a known niche component. By monitoring indi- vidual HSC behaviour using real-time imaging, we found that a portion of the homed HSCs underwent active division in the irra- diated mice, coinciding with their expansion as measured by flow assay. Thus, in contrast to central marrow, the endosteum formed a special zone, which normally maintains HSCs but promotes their expansion in response to bone marrow damage. To study the functional interaction between the HSC and its niche, we developed a method of ex vivo imaging stem cells (EVISC) (Fig. 1a and Supplementary Fig. 1). This method combines real-time imaging technology8 (Supplementary Movie 1) and the ability of HSCs to home to their niche after transplantation into irradiated mice9,10 (Supplementary Methods). To identify and track the transplanted HSCs, we isolated Flk22Lin2Sca-11c-Kit1 (Flk22LSK) cells that are enriched with purified HSCs9 from actin-driven GFP-transgenic mice (Supplementary Fig. 1a, b). We compared the behaviour of these cells after transplantation into irradiated and non-irradiated mice using two-photon microscopy11,12 (Fig. 1a, b). It has been docu- mented that HSCs migrate from tail vein to bone marrow in a few hours13,14. We obtained real-time images of HSC arrival in BM around 5���8 h after transplantation. In non-irradiated mice, we detected GFP1 HSCs in bone marrow (Fig. 1b and Supplementary Movie 2). In irradiated mice, however, we more frequently observed GFP1 HSCs homing to the endosteal region (Fig. 1c and Supplementary Movie 3). Our real-time imaging method enabled observation of dynamic interactions between HSCs and their niches. As the EVISC assay targeted limited areas, we obtained a more complete picture of the distribution of the transplanted GFP1 HSCs by immunostaining multiple longitudinal sections of femurs and tibias from three experiments. Positions of GFP1 HSCs were mea- sured as cell distance (where 1 cell distance (CD) 5 6���8 mm) from the endosteal surface. (Fig. 1d, e). We generated a cumulative percentage plot to display distribution patterns under irradiated versus non-irra- diated conditions (Fig. 1f). In non-irradiated recipients, GFP1 HSCs accumulated steadily with increasing distance from the endosteum. In contrast,in irradiatedrecipients, theGFP1 HSCs accumulatedrapidly close to the endosteum, but more gradually from 3 CD outwards. Two-sided Fisher���s exact test confirmed that the cells were signifi- cantly more likely to be located within #2 CD under irradiated than non-irradiated conditions (58.1% versus 12.5% respectively, P 5 0.009, Fig. 1g and Supplementary Table 1). Cell distribution pat- terns 3 CD from the endosteum and beyond were not significantly different between the two conditions (P 5 0.9576). In addition, we observed substantial reduction in bone marrow cellularity and enlarged blood vessels in irradiated bone marrow (Fig. 1d, e). Thus, after bone marrow damage, HSCs showed biased homing favouring the ���endosteal zone��� (# 2 CD) rather than central marrow ($3 CD). We also observed that GFP1 HSCs homed predominantly to the trabecular bone area (83.8%) compared with the compact bone area (16.2%) (P 5 0.001) under irradiated conditions, but showed no preference to either trabecular or compact bone area (42.3% com- pared with 57.7%) under non-irradiated conditions (Fig. 1h and Supplementary Table 2). We wondered what would be the mech- anism underlying the biased HSC homing, and found that expression of Sdf1 (ref. 15), a key chemotactic factor, was increased (2.7-fold) in trabecular bone area in response to irradiation (Fig. 1i). To confirm the location of HSCs in live mice, we used Scl-TVA transgenic mice in which an avian retrovirus receptor is driven from Scl-promoter-39enhancer regulatory elements that are active predomi- nantly, but not exclusively, in HSCs16,17. When these mice were injected with an avian virus containing a luciferase reporter (RCAS-Luc), only the Scl-TVA1 cells were susceptible to infection, allowing this popu- lationtobevisualizedbylive-imagingbioluminescence,whichreflected the luciferase activity7,18 (Supplementary Fig. 2). Bioluminescence imaging displayed only transient signals in liver and spleen (comparing 13 days with 4 months in Fig. 1j), reflecting short-term mobilization and potential expansion of HSCs in response to bone marrow damage, but showed strong and persistent signals (.10 months) in the trabecu- lar bone area of both legs and other regions (Fig. 1j, k and Supplementary Fig. 2c), reflecting self-renewing HSCs. The tendency of HSCs to home to the endosteum suggested pref- erential homing to the osteoblastic niche, which was thought to be 1 Stowers Institute for Medical Research, 1000 E. 50th Street, Kansas City, Missouri 64110, USA. 2 Department of Cardiology, Shanghai Rui Jin Hospital, Shanghai Jiao Tong University School of Medicine, 197, Rui Jin 2 Road, Shanghai 200025, China. 3Department of Microbiology and Immunology, and Greenebaum Cancer Center, University of Maryland School of Medicine, Baltimore, Maryland 21201, USA. 4Department of Pathology and Laboratory Medicine, Kansas University Medical Center, 3901 Rainbow Boulevard, Kansas City, Kansas 66160, USA. *These authors contributed equally to this work. Vol 457|1 January 2009|doi:10.1038/nature07639 97 Macmillan Publishers Limited. All rights reserved ��2009

surface or adjacent to Osx1 osteoblasts (Fig. 2k). This distribution pattern of N-cadherin1 cells adjacent to Osx1 cells suggested that N-cadherin1 cells were more probably pre-osteoblastic cells. We also validated the relationship between N-cadherin1 cells and osteoblasts (Fig. 2l, m) through staining sections derived from a Col2.3-GFP1 transgenic mouse in which GFP was driven by the collagen-Ia2.3 promoter and specifically expressed in osteoblasts22 (Fig. 2l). The endosteum offers homing or resident HSCs a range of possible microenvironments as discussed above. To determine with which type of cells GFP1 HSCs tended to interact, we co-stained with N-cadherin and GFP. GFP1 HSCs were observed directly attaching to N-cadherin1 cells (Fig. 2n, o, and Supplementary Fig. 5a) and were close or adjacent to such cells in 10 out of 17 cases (59% Supplementary Fig. 5a, b). This is consistent with our previous obser- vation of endogenous HSCs (identified using CD201 (ref. 24)) inter- acting with N-cadherin1 osteoblastic cells2,20. Notably, some of the GFP1 HSCs were also adjacent to N-cadherin1 cells in central marrow (Supplementary Fig. 5c). However, not all GFP1 HSCs were close to N-cadherin1 cells (Supplementary Fig. 5b), indicating the existence of additional N-cadherin2 niche components. Although the level of N-cadherin measured as fluorescence intens- ity in the GFP1 HSCs was much lower (average ninefold) compared to adjacent osteoblastic cells (Fig. 2p, q and Supplementary Table 5), its asymmetric distribution at the interface between HSCs and pre- osteoblastic cells (Fig. 2p and Supplementary Movie 5) validates our previous observation2. HSCs normally maintain a steady number but this can be increased in response to bone marrow damage25. We compared proliferation of homed GFP1 HSCs 4���6 h after transplantation using an immuno- assay with a marker Ki67 (Fig. 3a���d). In the non-irradiated bone marrow, clusters of proliferating cells were typically seen in the central marrow, and isolated proliferating cells were often seen in the endosteum (Supplementary Fig. 6a). In the irradiated host, how- ever, proliferating cells were substantially increased in the endosteal region (Supplementary Fig. 6b). Furthermore, we found that GFP1 HSCs were rarely Ki671 in non-irradiated hosts but were five times more often Ki671 in irradiated hosts (7% compared with 35%) (Fig. 3a���c and Supplementary Table 6). Notably, Ki671GFP1 HSCs were frequently found in locations adjacent to clusters of pro- liferating cells, and this was particularly obvious under irradiated conditions (Fig. 3b). Irradiation-induced changes in the location of proliferating cells were also reflected by more Ki671GFP1 HSCs but fewer Ki672GFP1 HSCs being detected in the endosteal compared to the central marrow regions (Fig. 3d and Supplementary Table 7). All these observations indicate that irradiation induced a dynamic change in the endosteal microenvironment from normal inhibition to stress-induced stimulation of proliferation, favouring the expan- sion of homed GFP1 HSCs. Using EVISC, we were also able to observe directly HSC prolifera- tion. We performed this experiment at different time points. At 5���8 h after transplantation (Fig. 3e), a GFP1 HSC located at the deepest part of a recess in bone gave rise to two daughter cells (Fig. 3f���i and Supplementary Movie 6). In another case, we observed two homed HSCs in the same field of view with different behaviours over 15-h real-time imaging: one was stable and the other was actively mobile and dividing (Fig. 3j, k and Supplementary Movie 8). At 36���40 h after transplantation (Supplementary Movie 7), three out of four GFP1 HSCs in the same area were stable at the endosteal region. We recorded active division of homed GFP1 HSCs in three out of twenty-one cases (14.2%) in three independent experiments. Our immunoassays identified the endosteal zone of irradiated recipients as a site of increased HSC proliferation after transplantation. Our Trabecular bone (TB) Diaphysis Epiphysis Metaphysis Compact bone (CB) 23% 13% 34% 30% 45% 40% 10% 0 20 40 60 80 100 CD31 only 6% N-cad only CD31 + N-cad Neither TB surface CB surface 6% g ���4 ���2 0 2 4 6 8 10 12 14 TBA CBA Coverage of CD31 + N-cad (%) (observed ��� expected) P=0.00004 P=0.47 Growth plate CD31 Compact bone Endosteum BM a Coverage (%) Coverage (%) 57% 47% 85% 45% 0 20 40 60 80 100 Total CD31 Total N-cad h i CB BM TB N-cad b CD31 only BM TB CD31N-cad c N-cad only CD31N-cad BM TB d N-cad CD31 CD31 + N-cad e TB BM CD31N-cad CB BM f 0 2 4 6 8 10 12 Pre- osteoblasts HSCs N-cad expression (��10 5 ) n = 4 P = 0.0025 q p Bone BM Highlight R+G BM BM Bone N-cad TB BM l Col2.3 ���GFP (green) Osx TB N-cad j k n m TB Osx (green) GFP���HSC o N-cad TB TB BM GFP���HSC N-cad BM Bone BM Periosteum Figure 2 | Relationships between osteoblastic and vascular structures and between GFP1 HSCs and osteoblasts. a, b, Distribution of vasculature (CD31, red a) and N-cadherin1 cells (N-cad, green b) in bone (DIC, grey) and bone marrow (DAPI, blue). c���f, Representative images of endosteal surface covered by CD31 only (c), N-cadherin only (d), and both (e) in the trabecular bone (TB) area and compact bone (CB) area (f). g, h, Percentage coverage by CD31 and N-cadherin (g), and quantification of the percentage coverage by CD31 only, N-cadherin only, both and neither (h see also Supplementary Table 3). The data were based on fluorescent signal intensity within 16 mm of endosteal surface. i, Statistic analysis of co-association between N-cadherin1 cells and vasculature (Supplementary Table 3). j���l, Co-staining Osx (j) with N-cad (k) and with GFP (l). m, Co- staining GFP with N-cad. n, o, A GFP1 HSC attaches to N-cadherin1 pre-osteoblastic cells. p, Three-dimensional view of cell in o showing asymmetric N-cadherin distribution in GFP1 HSC. q, N-cadherin expression (fluorescent intensity) in pre-osteoblasts versus GFP1 HSCs (average from four GFP1 HSCs). All error bars indicate s.d. NATURE|Vol 457|1 January 2009 LETTERS 99 Macmillan Publishers Limited. All rights reserved ��2009