Background correction and signal normalization was performed using the standard multichip average algorithm (54C55). Supplementary Material Supporting Information: Click here to view. Acknowledgments We thank Seth Karten for his kind help in proofreading the manuscript, Libuse Jerabek and Terry Storm for laboratory management, and Adrianne Mosley for help in animal work. (HSC) renewal in vitro, the other stromal populations promote HSC UNC0379 differentiation to more committed lines of hematopoiesis, such as the B-cell lineage. Gene expression analysis and microscopic studies further reveal a microenvironment in which CD105-, Thy1-, and 6C3-expressing marrow stroma collaborate to provide cytokine signaling to HSCs and more committed hematopoietic progenitors. As a result, within the context of bone as a blood-forming organ, the BCSP plays a critical role in supporting hematopoiesis through its generation of diverse osteogenic and hematopoietic-promoting stroma, including HSC supportive 6C3(+) niche cells. and and (a-2) isolated from actin-GFP mice. (Scale bar, 20 m.) (showing spongy bone marrow stroma derived from transplantation of population a-3. (Scale bar, 50 m.) (and and and and and and shows positive cells (red) with fibroblast morphology. (and after immunostaining with anti-osteocalcin antibody. Upper arrow points to a GFP(?) osteocalcin(+) individual osteocyte in cortical UNC0379 bone (red). Lower arrow points to GFP(+), osteocalcin(+) UNC0379 osteocyte (yellow). (showing GFP-labeled stromal cells (arrowheads). (Scale bar, 20 m.) (showing a chondrocyte cluster (forked arrowheads). (Scale bar, 20 m.) (after immunostaining with anti-collagen-2 antibody shows GFP(+), collagen2(+) chondrocytes (green and yellow). (Scale bar, 20 m.) To determine whether the CD105+Thy1?6C3? bone, cartilage, stromal progenitor (BCSP) subset is a heterogeneous population of separate chondrocyte-restricted and osteogenic-restricted progenitors, we assayed the differentiation potential of single BCSPs. Our data indicate that single BCSPs are multipotent and capable, at the single-cell level, of generating in vitro colonies containing collagen type 2-expressing chondrocytes and osteocalcin-expressing osteoblasts (Fig. 2 and and and < 0.05 by ANOVA; **< 0.0001 by ANOVA. We next explored the mechanisms by which 6C3+ stroma affect the endogenous HSC niche. To determine whether direct interaction with 6C3+ stroma facilitates HSC engraftment, we transplanted RFP-labeled HSCs and studied their association with stromal cells in situ by immunofluorescence (additional details in and for full materials and methods. Mice. C57BL/Ka-Thy1.1-CD45.1, C57BL/Ka-Thy1.1-CD45.1 (BA), C57BL/Ka-Thy1.2-CD45.1, and C57BL/Ka-Thy1.2-CD45.1- actin GFP, and rosa26-mRFP (C57BL/6[B6]) strains were derived and maintained in our laboratory. All animals were Rabbit polyclonal to ACBD5 maintained in Stanford University Laboratory Animal Facility in accordance with Stanford Animal Care and Use Committee and National Institutes of Health guidelines. Isolation and Transplantation of Adult and Fetal Skeletal Progenitors. Fetal skeletal elements (humerus, radius, tibia, femur, and pelvis) were dissected from C57/BL6 BA strain fetuses and digested in collagenase with DNase UNC0379 at 37 C for 40 min under constant agitation. Sorted and unsorted skeletal progenitors were pelleted and resuspended in 2 mL matrigel (regular) and then injected underneath the renal capsule of 8- to 12-wk-old anesthetized mice. HSC-Stromal Coculture and Transplant. To establish stromal populations for HSC coculture experiments, 200,000 CD105+Thy1-6C3-CD45-Tie2-alphaV+ BCSPs were fresh-sorted from dissociated bone and bone marrow stroma of e15.5 dpc, e17.5 dpc, or newborn (postnatal day 0C3) mice and plated on 0.1% (vol/vol) UNC0379 gelatin-coated 15-cm culture dish and supported with MEMalpha medium supplemented with 20% FCS and PenStrep (Invitrogen) antibiotic. Two weeks after culture, cells were lifted by incubating with M199 medium supplemented with Collagenase II at 1 mg/mL and then stained and FACs-sorted for indicated populations. Then 250 fresh-sorted HSCs were added to the stromal cells and cocultured in StemPro serum-free stem cell culture media supplemented with 10 ng/mL mouse recombinant steel factor (Peprotech), 5 ng/mL mouse recombinant Thrombopoietin (Peprotech), 20 ng/mL basic fibroblast growth factor (R&D), and 25 ng/mL insulin growth factor (R&D). Culture medium was changed by removing and replacing half of it with fresh medium every other day for 10 d. On the tenth day, cells were removed for analysis and or transplanted to irradiated mice. For HSC transplants, the contents of each well corresponding to 250 plated HSCs were combined with 300,000 unsorted host bone marrow cells as helper marrow and injected retro-orbitally into lethally irradiated (800 rad) congenic mice. Engraftment was assessed by FACS analysis of tail blood.