The procedures for bone and bone marrow section preparation, immunostaining conditions and antibodies are described in Supplementary Methods. The procedure for BrdU pulse labeling, LTR and subsequent detection has been reported 16 . The mice were fed BrdU (0.8 mg ml 21 in water) for 10 days, during which time 40% of LT-HSCs would divide at least once 31 . Seventy days after BrdU labelling, sections were stained with anti-BrdU antibody. N-cadherin 1 cell countFor quantitative analysis of N-cadherin þ cells, the sections were developed with AEC after being incubated with rabbit anti-N-cadherin antibody for 1 h and horseradish peroxidase (HRP)-conjugated goat anti-rabbit second antibody for 1 h. Three people counted the SNO cells in these sections, blind to the source of the sections. X-ray imageHigh-resolution X-rays (Faxitron MX-20) of bone and bone histomorphometry (OsteoMetrics, Inc.) were performed at the University of Missouri-Kansas City School of Dentistry. 1965-1972 (1996). 193-197 (2000). 10. Simmons, P., Gronthos, S. & Zannettino, A. C. Stem cell fate is influenced by specialized microenvironments that remain poorly defined in mammals 1-3 . To explore the possibility that haematopoietic stem cells derive regulatory information from bone, accounting for the localization of haematopoiesis in bone marrow, we assessed mice that were genetically altered to produce osteoblast-specific, activated PTH/PTHrP receptors (PPRs) 4 . Here we show that PPRstimulated osteoblastic cells that are increased in number produce high levels of the Notch ligand jagged 1 and support an increase in the number of haematopoietic stem cells with evidence of Notch1 activation in vivo. Furthermore, liganddependent activation of PPR with parathyroid hormone (PTH) increased the number of osteoblasts in stromal cultures, and augmented ex vivo primitive haematopoietic cell growth that was abrogated by g-secretase inhibition of Notch activation. An increase in the number of stem cells was observed in wild-type animals after PTH injection, and survival after bone marrow transplantation was markedly improved. Therefore, osteoblastic cells are a regulatory component of the haematopoietic stem cell niche in vivo that influences stem cell function through Notch activation. Niche constituent cells or signalling pathways provide pharmacological targets with therapeutic potential for stem-cell-based therapies.Mammalian bone marrow architecture involves haematopoietic stem cells (HSCs) in close proximity to the endosteal surfaces 5,6 , with more differentiated cells arranged in a loosely graduated fashion as the central longitudinal axis of the bone is approached 5,7,8 . This nonrandom organization of the marrow suggests a possible relationship between HSCs and osteoblasts-osteogenic cells lining the endosteal surface. Osteoblasts produce haematopoietic growth factors [9][10][11] and are activated by parathyroid hormone (PTH) or the locally produced PTH-related protein (PTHrP), through the PTH/ PTHrP receptor (PPR). We tested whether osteoblast...

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Microenvironmental signals can determine hematopoietic stem cell (HSC) fate choices both directly and through stimulation of niche cells. In the bone marrow, prostaglandin E 2 (PGE 2 ) is known to affect both osteoblasts and osteoclasts, whereas in vitro it expands HSCs and affects differentiation of hematopoietic progenitors. We hypothesized that in vivo

Hematopoietic stem cell (HSC) regulation is highly dependent on interactions with the marrow microenvironment. Controversy exists on N-cadherin's role in support of HSCs. Specifically, it is unknown whether microenvironmental N-cadherin is required for normal marrow microarchitecture and for hematopoiesis. To determine whether osteoblastic N-cadherin is required for HSC regulation, we used a genetic murine model in which deletion of IntroductionThe molecular signals that mediate regulatory microenvironmental osteoblastic-hematopoietic interactions provide potential therapeutic targets for hematopoietic stem cell (HSC) manipulation 1 but are still largely unknown. Based on the essential role of cadherins in fate specification of germline stem cells 2 and the close proximity of N-cadherin-expressing osteoblastic cells to HSCs, [3][4][5][6] it was initially proposed that N-cadherin may provide HSCs with instructive interactions with their niche. However, much debate was subsequently raised by reports questioning N-cadherin expression by HSCs, 7 whereas others suggested that different N-cadherin levels characterize functionally distinct HSC populations, 5,8,9 which may respond to niche manipulation. 10 Moreover, inducible global genetic N-cadherin deletion lacks hematopoietic defects, 11 but knockdown of N-cadherin in HSCs suppresses their long-term engraftment. 12 Whereas in HSCs N-cadherin, when present, is expressed at very low levels, 13 osteoblastic cells express N-cadherin at several differentiation stages, both in immature and mature cells. 3,14 Indeed, whereas germline deletion of the gene encoding N-cadherin (Cdh2) is embryonically lethal, 15 hemizygous mice already display osteoblastic defects, having accentuated bone loss with ovariectomy. 16 Moreover, recent data have suggested differential roles of N-cadherin at various stages of osteoblastic differentiation, with actions of N-cadherin both on osteogenic commitment as well as at terminal differentiation. 14 In this work, our goal was to determine whether targeted deletion of Cdh2 in maturing osteoblasts alters the BM microarchitecture, HSC number, and function in homeostasis and disrupts the action of parathyroid hormone (PTH) on the skeleton and/or on hematopoietic stem and progenitor cells (HSPCs). Methods MiceCol2.3-Cre mice expressing the Cre-recombinase under the control of the 2.3-kb fragment of the ␣1(I) collagen gene promoter were kindly provided by Dr Gerard Karsenty. 17 Mating of Col2.3-Cre with Cdh2 fl mice (generated in Dr Glenn L. Radice's laboratory, Thomas Jefferson University, Philadelphia, PA) 18 resulted in mice carrying the Col2.3-Cre ϩ Cadh2 fl/fl genotype, which had specific inactivation of N-cadherin in cells of the osteoblastic lineage, designated as OB-NCadh for the rest of the manuscript. 14 This line was maintained in the C57bl/6 background, and expression of the CD45.2 congenic marker was confirmed. Wild-type (WT) mice expressing the CD45.1 congenic marker (B6.SJL-Ptprc a Pep3 b /BoyJ CD45.1) were purchased from The Jackson L...