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STEM_780_sm_supplFig1a.pdf494KSupplementary figure 1a. Expression of αSMAcherry in bone and bone marrow. A high magnification image of a whole bone section allows for a more detailed and representative evaluation of the transgene expression. (a) Epifluorescence image. (b) Image following hematoxyllin counterstaining. Arrowheads indicate αSMAcherry+ cells encircling microavasculature within bone marrow and periosteum, while arrows indicate fibroblastic shaped cells expressing αSMAcherry within periosteum.
STEM_780_sm_supplFig1b.pdf527KSupplementary figure 1b. Differentiation of αSMAcherry+ cells into adipocyte lineage. (a) Bone marrow stromal cells derived from dual transgenic mice αSMAcherry/AP2cyan were imaged using brightfield and epifluorescence. αSMAcherry clearly identifies a population of fibroblastic cells forming colonies by day 7 while AP2cyan is active only in a few cells. (b) Proportion of the αSMAcherry expressing cells (left panel) and purity after sorts for αSMAcherry negative (middle panel) and sorted αSMAcherry+ cells (right panel). (c) Sorted αSMAcherry+ and (d) αSMAcherry– cells were replated and imaged 7 days following sorting. The αSMAcherry+ cells activated the adipocytic marker AP2cyan while αSMAcherry– did not show cyan expression. (e) Gene expression assessed by real-time PCR in cultures derived from unsorted (preSORT) and sorted/replated αSMAcherry+ (SORTpos) and αSMAcherry– (SORTneg) cells following induction with adipogenic media. (f) Oil red O staining detects adipocytic vacuoles in αSMAcherry+ cells while no Oil red O staining is detected in αSMAcherry– cells (g).
STEM_780_sm_supplFig2a.pdf799KSupplementary figure 2a. In vivo lineage tracing differentiation using SMA9 transgenic mice. High power scans showing SMA9 expression patterns in (a) SMA9/Untreated, (b) SMA9/Tamoxifen treated; sacrificed 2 days after treatment, (c) SMA9/Tamoxifen treated; sacrificed 17 days after treatment. Very few or no SMA9 labeled cells are observed in the nontreated mice. However, following tamoxifen injections a population of cells in the trabecular area and within the periosteum show SMA9 expression. This population can be traced into numerous osteoblast and osteocytes 17 days after the termination of the tamoxifen treatment GP- growth plate.
STEM_780_sm_supplFig2b.pdf221KSupplementary figure 2b. Activation of αSMACreERT2 transgene in soft tissues. The activation of αSMACreERT2 transgene with or without tamoxifen induction, was evaluated in several tissues with high levels of expression of endogenous αSMA gene. The SMA9 expression was detected after induction with tamoxifen in lung and liver (a-b, right panels) in cells associated with vasculature, whereas no SMA9 signal was observed in tissues from mice that were not treated with tamoxifen (a-b, left panels). The presence of SMA9 expressing cells was detected in both tamoxifen treated (c-d, right panels) and untreated (c-d, left panels) aorta and bladder, indicative of the leakiness of Cre activation in tissues in which αSMA promoter exhibits strong activity.
STEM_780_sm_supplFig2c.pdf176KSupplementary figure 2c. In vitro activation of αSMACreERT2 transgene. Primary bone marrow stromal cell cultures were established from 3 experimental groups, to evaluate the in vitro activation of the transgene by tamoxifen; (a, d) αSMACreERT2negative/Ai9/treated, (b, e) SMA9/untreated and (c, f) SMA9/treated. Cells were enzymatically digested and analyzed by FACS to evaluate the proportion of αSMACre induced tdTomato activation on day 3 (a-c) and day 7 (d-f) of the cultures.
STEM_780_sm_supplFig3a.pdf269KSupplementary figure 3. Surface phenotype profiling of bone marrow and periosteal SMA9-labeled cells. (a) FACS analysis of the percentage of SMA9+ cells in the bone marrow and periosteal cell compartments, 2 and 17 days following in vivo tamoxifen treatment. Results for αSMACreERT2negative/Ai9/treated (left panels) and SMA9/treated (right panels) are shown. Each group included 2-3 independent samples pooled from the respective tissue of 2-3 mice. To determine the percentage of tdTomato+ cells at least 2.5×106 cells for bone marrow and 0.25×106 for periosteal cells were analyzed. Gates were set using control populations from tamoxifen injected αSMACreERT2negative/Ai9 mice. Mice without tamoxifen treatment, carrying the alphaSMACreERT2 transgene, served for the Cre-leakage control and contained the percentage of tdTomato+ cells comparable to the control mice (not shown). (b) Dot-plots represent the populations expressing respective mesenchymal cell marker against hematopoietic markers within the SMA9+ cell compartment. FACS analysis was performed by gating SMA9+ cells and plotting them using hematopietic markers (CD45/Ter119/CD11b) vs. respective proposed mesenchymal lineage marker (Sca-1, CD90, CD31, CD51 Cd140b, CD146 or CD106) within bone marrow and periosteal cell populations. Lineage marker expression was analyzed on gated SMA9+ cells using at least 10000 gated cells for bone marrow and 5000 cells for periosteal cell populations. Gates were set using non-stained samples from the respective tissue (not shown).
STEM_780_sm_supplFig3b.pdf337KSupplementary figure 3. Surface phenotype profiling of bone marrow and periosteal SMA9-labeled cells. (a) FACS analysis of the percentage of SMA9+ cells in the bone marrow and periosteal cell compartments, 2 and 17 days following in vivo tamoxifen treatment. Results for αSMACreERT2negative/Ai9/treated (left panels) and SMA9/treated (right panels) are shown. Each group included 2-3 independent samples pooled from the respective tissue of 2-3 mice. To determine the percentage of tdTomato+ cells at least 2.5×106 cells for bone marrow and 0.25×106 for periosteal cells were analyzed. Gates were set using control populations from tamoxifen injected αSMACreERT2negative/Ai9 mice. Mice without tamoxifen treatment, carrying the alphaSMACreERT2 transgene, served for the Cre-leakage control and contained the percentage of tdTomato+ cells comparable to the control mice (not shown). (b) Dot-plots represent the populations expressing respective mesenchymal cell marker against hematopoietic markers within the SMA9+ cell compartment. FACS analysis was performed by gating SMA9+ cells and plotting them using hematopietic markers (CD45/Ter119/CD11b) vs. respective proposed mesenchymal lineage marker (Sca-1, CD90, CD31, CD51 Cd140b, CD146 or CD106) within bone marrow and periosteal cell populations. Lineage marker expression was analyzed on gated SMA9+ cells using at least 10000 gated cells for bone marrow and 5000 cells for periosteal cell populations. Gates were set using non-stained samples from the respective tissue (not shown).
STEM_780_sm_supplFig4.tif817KSupplementary figure 4. Surface phenotype profiling of SMA9-labeled primary bone marrow stromal cells. FACS analysis of the SMA9+ cells in the bone marrow stromal cell culture established from SMA9 tamoxifen treated mice. FACS analysis was performed by gating SMA9+ cells and plotting them using hematopoietic markers (CD45/Ter119) vs. respective proposed mesenchymal lineage markers (Sca1, CD51, CD140b, CD 146 or CD31). Gates were set in accord to the non-stained samples from the respective tissue (not shown). The expression profile for Sca1+ (a, d), CD51 (b, e) and CD140b (c, f) is presented for day 3 (ac) and day 7 (d-f) of the cultures. We found no expression of CD146 or CD31 (not shown).
STEM_780_sm_supplFig5.pdf120KSupplementary figure 5. Mesenchymal progenitor cell lineage tracing into chondrogenic phenotype during fracture healing. (a-c) Chondrogenesis during fracture healing is examined at one week following fracture. The expression of SMA9 is detected in the cells morphologically identified as chondrocytes within the callus area (a). One week after fracture the expression of chondrogenic marker Col2a1-cyan is detected in SMA9+ cells (b-c). The simultaneous expression of SMA9-red and Col2a1-cyan is indicated by arrows (a-b), and an overlayed image shows purple cells (see arrows). The interrupted line indicates the site of the fracture. Upper panel are images obtained at 10x magnification and lower panel at 20x magnification of the outlined area.
STEM_780_sm_supplMethods.pdf102KSupplementary data

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