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Keywords:

  • Adult stem cells;
  • Bone;
  • In vivo tracking;
  • Marrow stromal cells;
  • Mesenchymal stem cells

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSION
  8. Acknowledgements
  9. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  10. REFERENCES
  11. Supporting Information

Adult mesenchymal progenitor cells have enormous potential for use in regenerative medicine. However, the true identity of the progenitors in vivo and their progeny has not been precisely defined. We hypothesize that cells expressing a smooth muscle α-actin promoter (αSMA)-directed Cre transgene represent mesenchymal progenitors of adult bone tissue. By combining complementary colors in combination with transgenes activating at mature stages of the lineage, we characterized the phenotype and confirmed the ability of isolated αSMA+ cells to progress from a progenitor to fully mature state. In vivo lineage tracing experiments using a new bone formation model confirmed the osteogenic phenotype of αSMA+ cells. In vitro analysis of the in vivo-labeled SMA9+ cells supported their differentiation potential into mesenchymal lineages. Using a fracture-healing model, αSMA9+ cells served as a pool of fibrocartilage and skeletal progenitors. Confirmation of the transition of αSMA9+ progenitor cells to mature osteoblasts during fracture healing was assessed by activation of bone-specific Col2.3emd transgene. Our findings provide a novel in vivo identification of defined population of mesenchymal progenitor cells with active role in bone remodeling and regeneration. STEM CELLS 2012; 30:187–196.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSION
  8. Acknowledgements
  9. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  10. REFERENCES
  11. Supporting Information

A full understanding of the recruitment of multipotent progenitors into the skeletal lineage and the factors influencing their differentiation is critical for the development of protocols to modulate bone regeneration and to the design of novel targets for pharmacological intervention. However, a major obstacle has been the unavailability of reproducible methods to identify these progenitors and to track their fate. Initial studies have characterized some phenotypic properties of these cells but have not provided clear information on their origin.

A perivascular niche has been implicated as a source of mesenchymal progenitors [1, 2]. The human bone marrow-derived stromal population expressing STRO-1 and CD146 exhibits the ability to form bone in vitro and in a heterotopic bone formation assay in vivo. This population of cells is localized in the proximity of endothelial cells and was positive for αSMA expression [3]. Sacchetti et al. [4] defined a population of osteoprogenitors as CD146+ subendothelial cells in bone marrow suggesting that perivascular cells exhibit potential for self-renewal and differentiation supportive of hematopoiesis. Crisan et al. [5] observed perivascular cells in various organs with the ability to differentiate into multiple mesenchymal lineages.

Recently, we described a population of cells expressing smooth muscle actin α (αSMA) that have osteogenic potential [6]. The αSMAGFP cells are located in perivascular niches, periosteum and sutures, regions that contain osteoprogenitor cells. Moreover, αSMAGFP+ cells derived from primary bone marrow stromal cells (BMSCs) and adipose derived stromal cells of transgenic mice exhibit osteogenic and adipogenic potential [6]. A high proportion of αSMAGFP+ cells express the stem cell markers, stem cell antigen 1 (Sca1) and CD90, but lack expression of the CD117 (c-kit) or CD11b [7–9].

A detailed characterization of progenitor cells requires methods to isolate the cells and trace their transition into different phenotypes. The use of cell surface markers is limited by the fact that progenitor cells may lose markers as they differentiate.

We have generated αSMACreERT2 transgenic mice that provide a system in which the fate of progenitors can be traced. We propose to examine the ability of αSMACreERT2-expressing cells as mesenchymal progenitors during bone formation and repair and to determine whether these progenitor cells become committed to specific lineages. The combinatorial approach of using Cre recombinase fused to a modified estrogen receptor ligand binding domains (ERT2) permits the temporal induction by delivery of tamoxifen and tissue specificity by directing the Cre expression with a lineage-specific promoter [10]. When crossed with Ai9 reporter mice, recombination can be detected by expression of red fluorescence (TdTomato) [11]. This system allows for the spatial and temporal activation of Cre labeling, identification of the labeled population, and tracing the lineage progenies of the labeled cells for prolonged periods after the tamoxifen treatment has been terminated. Here, we used the visual markers for transgene expression and the Cre/loxP recombination system for lineage tracing using a fragment of the αSMA promoter [6]. We have used murine models harboring real-time visual transgenes in combination with cell surface markers and Cre/loxP technology as a powerful way of characterizing mesenchymal progenitors.

These approaches have defined a mesenchymal progenitor cell in vivo that actively participates in the physiological process of bone remodeling and during the regenerative processes of fracture healing.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSION
  8. Acknowledgements
  9. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  10. REFERENCES
  11. Supporting Information

Generation of Transgenic Mice and Animal Studies

Detailed description of generation of αSMACreERT2 mice is outlined in the Supporting Information Methods. The generation and characterization of αSMAGFP, Col2.3cyan, Col2a1cyan, and Col2.3emd mice have been published [6, 12, 13]. Mice transgenic for AP2cyan and αSMAcherry have been recently developed [14]. The αSMACreERT2/Ai9 dual transgenic mice (term SMA9 will be used) were generated by breeding αSMACreERT2 male mice and Ai9 female mice that were obtained from Jackson Labs (Bar Harbor, ME stock # 007905, jaxmice.jax.org). The Ai9 mice harbor a targeted mutation of the Gt(ROSA)26Sor locus with a loxP-flanked STOP cassette preventing transcription of a CAG promoter-driven red fluorescent protein variant (tdTomato). For in vivo lineage tracing studies, 4–5-week-old mice were treated with tamoxifen at the dose of 75 μg/g of weight. Mice were treated twice in the interval of 24 hours and sacrificed 2 days or 17 days later. The SMA9-untreated mice and αSMACreERT2negative/Ai9-treated mice were evaluated as controls.

In Vitro Studies

Primary BMSCs were prepared as previously described [15]. Following cell separation using fluorescence-activated cell sorting (FACS), cells were plated at the density of 10,000 cells per centimeter square in 24-well plates. Cells were induced to osteogenesis using Dulbecco's modified Eagle's medium/10% fetal calf serum (FCS) medium supplemented with 50 μg/ml ascorbic and 8 mM β-glycerol phosphate for 2 weeks, while adipogenesis was induced using media supplemented with 1 μM insulin and 0.5 μM rosiglitasone for 7 days. To evaluate chondrogenesis, sorted SMA9+ and SMA9 population were seeded as a 20 μl spot containing 5 × 104 cells. After 2 hours, α minimal essential medium (MEM)/10% FCS was added, and 24 hours later cells were placed under chondrogenic induction (serum-free media supplemented with 50 μg/ml ascorbic acid, 10 ng/ml transforming growth factor (TGF)β3, 100 nM dexamethasone, 40 μg/ml L-proline, sodium pyruvate, ITS+ 1 (Sigma, St. Louis, MO, www.sigmaaldrich.com) and cultured in 5% oxygen) for 7 days. Methods evaluating osteogenesis, adipogenesis, and chondrogenesis in vitro are presented within Supporting Information Methods section.

Histology

Bone samples for histology were fixed for 3–5 days in 10% formalin (Sigma), decalcified in 15% EDTA for 4–7 days depending on the age of animal, placed in sucrose overnight, and embedded in cryomedium (Thermo Fisher Scientific (Waltham, MA, www.thermofisher.com)). Soft tissues were fixed in 10% formalin overnight, transferred to sucrose, and after 24 hours they were embedded and sectioned. Sections of 5 μm were obtained using a Leica (Wetzler, Germany, www.leica-microsystems.com) cryostat and tape transfer system (Section-lab, Hiroshima, Japan, section-lab.jp). Images were obtained using appropriate filter cubes optimized for green fluorescent protein (GFP) variants (Chroma Technology, Bellows Falls, VT, www.chroma.com) using a Observer.Z1 microscope (Carl Zeiss, Thornwood, NY, www.zeiss.com). Images were obtained in gray scale, pseudocolored, and composite images were assembled. To obtain a full-size image of bone, images were scanned at high power and then stitched into a composite. Following fluorescent imaging, sections were stained with hematoxylin and imaged. Cells embedded within the bone matrix were counted within the trabecular area using sections counterstained with 4′,6-diamidino-2-phenylindole (DAPI) to visualize the population of osteocytes.

Flow Cytometry

FACS analysis and cell sorting of αSMAcherry+ and αSMACreERT2/Ai9 cells were performed using a BD FACSAria I (BD Biosciences, San Jose, CA, www.bdbiosciences.com) equipped with five lasers and 18 fluorescence detectors. Flushed bone marrow cells and enzymatically digested (collagenase 0.2%, hyalouronidase 0.2% in 0.25% trypsin) periosteal layer cells were used. Erythrocytes were lysed by hypotonic shock. Sorting gates were defined using cells from nontransgenic and transgenic mice bearing individual transgenes. Single-cell suspensions were prepared in staining medium (2% FCS/Hanks' balanced saline solution/HEPES). For phenotype characterization, we used commercially available antibodies (Ab) as described in detail in Supporting Information Material.

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSION
  8. Acknowledgements
  9. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  10. REFERENCES
  11. Supporting Information

αSMA+ Cells Exhibit Characteristics of Mesenchymal Progenitor Cells

To evaluate the localization of cells expressing αSMA, we analyzed mice expressing cherry fluorescent protein under the control of αSMA promoter. In bone marrow and periosteum, we observed αSMAcherry+ cells in association with microvasculature. In addition to perivascular location, we observed αSMAcherry+ fibroblastic-shaped cells in periosteum (Fig. 1A–1D, Supporting Information Fig. 1a for whole bone section). FACS analyses of freshly isolated cells indicated that the αSMA transgene is active in 0.02%–0.03% of cells in bone marrow and in around 1% of scraped cells from periosteum (Fig. 1E–1H). The identified population in bone marrow can be expanded in vitro by day 7 (Fig. 1I). To assess the ability to differentiate into osteogenic lineage, αSMAcherry+ and αSMAcherry population were sorted from a dual transgenic mouse harboring the osteoblast specific reporter, Col2.3cyan (Fig. 1J). These cells were then replated and grown under lineage-inductive conditions. We confirmed that αSMAcherry+ cells differentiated into mature osteoblasts and expressed Col2.3cyan on days 7 (Fig. 1L) and 14 (Fig. 1N). In contrast, αSMAcherry cells did not generate colonies or express the osteogenic marker (Fig. 1K, 1M). The phenotype of cells derived from the αSMAcherry+ population was confirmed by staining for alkaline phosphatase and mineralization and the expression of bone sialoprotein and osteocalcin (Fig. 1O, 1P). Similar experiments driving sorted cells into adipogenesis and chondrogenesis showed that the αSMAcherry+ population has the ability to differentiate into mature adipocytes and chondrocytes. Adipogenesis was detected by staining for oil red O and by expression of AP2cyan and mRNA for adipsin and adiponectin (Supporting Information Fig. 1b), while chondrogenic differentiation was analyzed by alcian blue staining and aggrecan mRNA expression (data not shown).

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Figure 1. αSMAcherry-expressing cells display osteoprogenitor potential. (A–D): The expression of αSMAcherry is localized to the perivascular area of capillaries within periosteum and muscle (A, B, see arrows) and bone marrow (C, D, arrowheads). (E–H): Fluorescence-activated cell sorting (FACS) analysis for αSMAcherry expression in samples derived from enzymatically digested periosteum (E, F) or whole bone marrow from transgenic (G, H) or nontransgenic mice (E, G). Proportion of αSMAcherry-expressing cells is indicated in the right lower corner of the plots (four αSMAcherry mice were analyzed individually and the representative values are shown). (I, J): Primary BMSC derived from αSMAcherry/Col2.3cyan mice were grown for 7 days and imaged under transmitted (left), red (middle), and blue (right) epifluorescence. αSMAcherry expression was present in fibroblastic-shaped cells while no expression of Col2.3cyan was detected (I). αSMAcherry-expressing cells (J, left panel, 23% of cells) were collected by FACS sorting; reanalysis of the sorted population purity is shown for αSMAcherry and αSMAcherry+ cells (J, middle and left panels). (K, L): Following sorting, cells were replated and induced to osteogenesis and FACS analyzed on day 7. αSMAcherry cells did not activate Col2.3cyan (K) while α-SMAcherry+ showed differentiation into Col2.3cyan+ cells (L). Col2.3cyan was detected at day 14 in population of differentiated α-SMAcherry+ cells (N), with no expression in α-SMAcherry population (M). (O, P): Alkaline phosphatase expression and mineralization (arrows) was detected in αSMAcherry+ cells in contrast to αSMAcherry cells (O). Real-time PCR shows increased expression of BSP and OC after osteogenic induction of α-SMAcherry+ cells (P). Abbreviations: BMSC, bone marrow stromal cells; PCR, polymerase chain reaction; SSC, side scatter; BSP, bone sialoprotein; OC, osteocalcin.

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Lineage Tracing Identifies Progenitor Population During Normal Skeletal Remodeling

To determine whether the αSMA promoter can identify cells with progenitor activity in vivo, we generated αSMACreERT2 transgenic mice and characterized its expression by crossing it with the Ai9 reporter transgenic line to generate αSMACreERT2/Ai9 (SMA9) mice (Fig. 2A). In these mice, the expression of the Cre molecule is activated under the control of the αSMA promoter upon treatment with tamoxifen. This allows the tracking of developmental progression of αSMA-expressing cells by activating the expression of the tdTomato visual reporter through Cre-mediated recombination. Mice were treated with tamoxifen and analyzed at days 2 and 17 after treatment (Fig. 2B–2D). After 2 days of treatment, SMA9+ cells defined a small population of cells present in primary spongiosa (Fig. 2C) or within the periosteum (Supporting Information Fig. 2a). No cells expressing SMA9 reporter were detected within the bone matrix (Fig. 2C). Interestingly, 17 days after the tamoxifen activation, we found numerous osteoblasts and osteocytes labeled by the transgene. Image analysis revealed that 23.4% ± 3.2% of trabecular bone matrix-embedded cells were SMA9+ at this time point. These observations confirm the ability of the αSMA-directed Cre to target osteoprogenitor cells in vivo (Fig. 2D). We have confirmed that the αSMACreERT2 activity is ligand dependent in adult bone and other tissues. In the absence of tamoxifen, we did not detect leaky expression of tdTomato in bone tissue (Supporting Information Fig. 2a). However, a significant leak of the Cre expression was observed in tissues with strong endogenous activity of αSMA gene like bladder and aorta (Supporting Information Fig. 2b).

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Figure 2. Cell lineage tracing experiments following in vivo transgene activation. (A): The αSMACreERT2 transgenic construct harboring sequence of smooth muscle actin α (αSMA) regulatory elements. These mice were crossed with a Cre recombinase reporter strain Ai9. In dual expressing SMA9 mice, tamoxifen-induced Cre recombinase activates expression of tdTomato. (B): Diagram of experimental approach for the cell lineage tracing experiments. Mice were sacrificed on days 2 and 17 following tamoxifen treatment and evaluated by histological analysis. Primary BMSCs were established from mice treated with tamoxifen and sacrificed on day 2 after treatment. (C, D): Histological analysis of SMA9 recombination identifies SMA9+ cells within the TR but not inside the bone matrix (C, arrowheads 2 days after treatment). No expression is observed in the GP chondrocytes. After 17 days of the initial evaluation (day 17), we could track the SMA9+ cells to osteoblasts lining trabecular bone (D, arrowheads) and ostecytes embedded within bone (arrows). Representative images of six to seven mice per group analyzed at each time point are shown. (E): Epifluorescent imaging of primary BMSC derived from SMA9 mice treated with tamoxifen. Once labeled in vivo, SMA9+ cells expanded during the first week in culture formed nodules and underwent mineralization (arrow). (F–K): FACS analysis of primary BMSC (days 3 (F–H) and 7 (J, K)) derived from SMA9+-labeled cells following tamoxifen injection in vivo was performed. αSMACreERT2negative/Ai9 mice treated with tamoxifen (F, I), and SMA9 mice without treatment (G, J) served as a controls. In vivo labeled SMA9+ cells account for approximately 1% of cells on day 3 and close to 25% on day 7 of the culture (H, K). Abbreviations: BMSC, bone marrow stromal cells; GP, growth plate; SSC, side scatter; TR, trabecular area.

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To further evaluate the distribution of labeled cells generated after αSMACre activation, we analyzed primary cultures at days 3 and 7 by flow cytometry. We did not detect expression of tdTomato in cells derived from αSMACrenegative/Ai9/Tx-treated mice (Fig. 2F, 2I) at days 3 or 7 or in cells derived from SMA9 untreated mice on day 3 in culture (Fig. 2G). However, an appreciable activation of Cre-directed recombination was observed in SMA9-treated mice by day 3 (1%–2%) and by day 7 (23%–27%; Fig. 2H, 2K). A small proportion of cells showed a spontaneous Cre activation in primary cultures derived from αSMA9 nontreated mice (Fig. 2J) by day 7. The spontaneous activation of the αSMACreERT2 transgene in vitro is due to the increase in αSMA promoter activity and the presence of serum response elements in the proximal sequence of the αSMA promoter [16].

In addition, primary cultures derived from untreated SMA9 mice, subsequently treated in vitro by 4-OH tamoxifen, showed expression of tdTomato in a similar proportion of cells (Supporting Information Fig. 2c). This confirms our in vivo observation that we are selectively targeting a progenitor population that exhibits the ability to differentiate into mature osteoblasts in vivo and in vitro.

Differentiation Potential of SMA9-Labeled Progenitor Population

To test the progenitor ability of the in vivo-labeled SMA9 cells, we established primary cultures from SMA9 mice that were treated with tamoxifen for 2 days (Fig. 2B). After 1 day in culture, individual SMA9+ cells were evident, and over time they expanded in number forming colonies during the first 7 days of culture (Fig. 2E). After induction of osteogenic differentiation, SMA9+ cells formed multilayered nodules with the ability to mineralize. To substantiate these results, we have used primary BMSC cultures derived from SMA9/AP2Cyan and SMA9/Col2.3emd mice treated with tamoxifen. Cultures were established 2 days after tamoxifen treatment and after reaching confluence were induced to adipogenesis and osteogenesis. We have observed numerous SMA+ cells differentiating into adipocytes or osteoblasts as detected by the expression of AP2cyan (Fig. 3B, 3C) or Col2.3emd, respectively (Fig. 3E, 3F). To evaluate chondrogenic differentiation, SMA9 mice were used, and cells were sorted into SMA9+ and SMA9 populations on day 7 of culture. After differentiation, we observed the formation of alcian blue positive nodules in the cultures derived from SMA9+ cells (Fig. 3I). Cultures of unsorted or SMA9 cells did not form alcian blue positive nodules (Fig. 3G, 3H) even after longer period in culture. The SMA9+ population shows also enhanced mRNA expression of chondrogenic markers Col2a1 and aggrecan (data not shown).

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Figure 3. Multilineage potential of SMA9 progenitor cells. (A–F): Primary BMSCs were established from SMA9/Col2.3emd and SMA9/AP2cyan mice treated with tamoxifen for 2 days. Cells were then grown to confluence and induced to osteogenic or adipogenic differentiation, respectively. Osteogenic differentiation was confirmed by dual expression of red-labeled SMA9 (B), and green-labeled Col2.3emd osteoblast lineage cells (C) (phase is shown in A). Adipogenic differentiation was confirmed by dual expression of red-labeled SMA9 (E), and blue-labeled AP2cyan adipocytes (F) (phase is shown in D). (G–I): To evaluate chondrogenesis, cells derived from SMA9 mice cells were cultured for 7 days and then sorted into SMA9+ and SMA9 and placed under chondrogenic conditions for 9 days. Formation of chondrogenic nodules was detected using alcian blue staining in sorted SMA9+ cells (I), in contrast to SMA (H) and unsorted BMSC (G). Abbreviation: BMSC, bone marrow stromal cells.

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Our data clearly show that the SMA9 transgenic constructs provided a bonafide identification of mesenchymal progenitor cells and should allow further characterization of this population of cells using markers currently proposed as signature molecules for stem cells.

Multiple Mesenchymal Stem Cell Markers Are Present on SMA9-Labeled Cells

In addition to the histological assessment, we followed the percentage of SMA9+-labeled cells in vivo at days 2 and 17 after tamoxifen treatment by flow cytometry. We found an increase in the percentage of cells expressing tdTomato reporter in the bone marrow up to 0.3% at day 17, compared to the 0.02% observed for the expression of αSMAcherry in basal conditions (Fig. 4A). In contrast, the percentage of SMA9+ cells isolated from the periosteal layer was approximately 1% and was maintained along a time course of evaluation (Fig. 4B, Supporting Information Fig. 3a). We used multicolor FACS analysis to test if the reporter signal correlated to markers of the hematopoietic lineage. To evaluate hematopoietic markers, we used a combination of antibodies for CD45, Ter119, and CD11b. Interestingly, most of the SMA9+ cells present in the bone marrow expressed markers characteristic of hematopoietic lineage, whereas the majority of the SMA9+ periosteal cells were (CD45/Ter119/CD11b) (Fig. 4A, 4B). Among the (CD45/Ter119/CD11b) SMA9+ bone marrow population, approximately one-third of the cells were CD45+Ter119CD11b (not shown). We also analyzed gated SMA9+ cells for the coexpression of cell surface markers associated with mesenchymal progenitor cells and found that SMA9+ population maintained a stable expression pattern when analyzed at days 2 and 17 after transgene induction (Fig. 4C, 4D, Supporting Information Fig. 3b). Bone marrow cells that are tdTomato+ and (CD45/Ter119/CD11b) were mostly negative for the included mesenchymal lineage markers, except for CD31 that was weakly expressed on approximately 5% cells on day 17 (Fig. 4C).

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Figure 4. Distinct phenotype of αSMACre-labeled cells from the BM compartment and periosteum. (A, B): Left-side graphs present fluorescence-activated cell sorting (FACS) analysis of the percentage of αSMAcherry- and SMA9-labeled cells (SMA9+) (2 and 17 days following tamoxifen injection) within the BM (A) and among periosteal cells (B). Right-side graphs present relative proportion of (CD45/Ter119/CD11b)+ versus (CD45/Ter119/CD11b) populations among αSMAcherry and SMA9+ cells. Each group included two to three independent samples pooled from the respective tissue of two to three mice. To determine the percentage of SMA9+ cells at least 2.5 × 106 cells for BM and 0.25 × 106 for periosteal cells were analyzed. Gates were set in accord to the control populations from tamoxifen-injected αSMACrenegative/Ai9 mice. Mice without tamoxifen treatment, carrying the transgene SMA9 served for the Cre-leakage control and contained a percentage of tdTomato+ cells comparable to the control mice (not shown). (C, D): Phenotyping of SMA9+ cells 2 and 17 days following transgene activation. Graphs represent the percentage of the population expressing respective mesenchymal cell markers within (CD45/Ter119/CD11b)+ versus (CD45/Ter119/CD11b) compartments of the SMA9+ cells. FACS analysis was performed by gating SMA9+ cells and plotting them using markers for hematopoietic lineage (CD45/Ter119/CD11b) versus respective proposed mesenchymal lineage marker (Sca1, CD90, CD31, CD51 Cd140b, CD146, or CD106) within the bone marrow (C) and periosteal cell populations (D). Values represent mean ± SD of the percentage of respective population from two to three independent samples pooled from two to three mice. Abbreviations: BM, bone marrow; SMA, smooth muscle actin.

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Percentages of hematopoietic marker positive SMA9+ bone marrow cells expressing mesenchymal markers vary between 10% and 30%, with CD31 being the most prominently expressed (Fig. 4C). This percentage of CD31+ cells was much lower (<0.1%) in αSMAcherry-labeled cells (data not shown), indicating induction of CD31 expression after tamoxifen activation of Cre mediated recombination.

The periosteal SMA9+ cells had a different phenotype, mostly lacking expression of hematopoietic markers but strongly expressing Sca1 and CD90 on approximately 20% of the population and weakly expressing CD51 on approximately a third of the population at day 2 after transgene induction (Fig. 4D, Supporting Information Fig. 3b).

The in vitro system allows for the expansion of the progenitor population but comes with a number of variables including serum composition and selective expansion of the adherent population of cells. Therefore, we evaluated the expression of cell surface markers that are used to characterize mesenchymal progenitor cells in vitro. Following our in vivo indication that the majority of SMA9+ bone marrow cells are positive for the hematopoietic lineage markers, we have evaluated the expression of SMA9+ cells for the presence of Sca1, CD51, CD140b, CD31, and CD146 in the context of CD45/Ter119 expression. During the first 3 days in culture, SMA9+ cells lose the expression of CD45/Ter119, which is present in approximately 2% of cells on day 3 and 6% on day 7 (Supporting Information Fig. 4). The expression of Sca1 and CD51 was observed by 30%–40% of cells on day 3 and increased up to 50%–60% of cells on day 7, while CD140b was expressed at much lower levels (Supporting Information Fig. 4). We did not observe the expression of CD31 or CD146 in primary stromal cell cultures at either time point (not shown).

Origin of Mesenchymal Lineages During Fracture Repair

To determine whether SMA9-expressing cells participate in regenerative processes including generation of chondrocytes and osteoblasts, we used a tibia fracture model. The experimental design included treatment with tamoxifen, on the day before and on the day of fracture. The aim was to label the population of αSMA-expressing cells and then trace their progeny during the fracture-healing process.

During the first week following injury, expansion of SMA9+ cells within bone marrow and periosteum was observed (Fig. 5A). Interestingly, we detected the presence of SMA9+-expressing cells in newly formed chondrogenic areas of the fracture callus and areas of the new bone formation (Fig. 5B). We correlated the presence of these osteoblasts with the dynamic formation of new bone through injection of calcein dye 1 day before sacrificing the mice. The osteoblasts derived from SMA9+ progenitor cells showed intense deposition of new bone, indicated by the correlation between calcein label (green) and tdTomato (red) positive cells (Fig. 5B). These results clearly indicated that the labeled populations present within the periosteum and bone marrow actively participate in all the aspects of fibrochondral ossification.

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Figure 5. Mesenchymal progenitor cell lineage tracing during fracture healing. (A, B): Osteogenesis during fracture healing is examined at 1 and 2 weeks following fracture. The expression of SMA9 is detected in the periosteal layer of cells (A, arrowheads point to the expanded SMA9+ cells within periosteum) located in proximity to the fracture site (indicated by perforated lines). After 1 week of fracture, SMA9+ cells comprise most of the periosteal layer around fracture site. BM is also filled with SMA9+ cells. After 2 weeks of the fracture, new cartilaginous callus formation is observed (B, arrowheads), while new bone formation is detected by deposition of calcein (green). Osteogenic lineage cells labeled by the SMA9 reporter (red, tdTomato+) persist in proximity to the large and intermingled calcein-labeled areas within the callus. (C): After 2 weeks of fracture, callus of a SMA9 control mouse (tamoxifen nontreated) shows extensive calcein labeling but minimal reporter leakage. (D, E): The same experimental design was completed in a SMA9/Col2.3emd model. The expression of SMA9+ cells (D, red) colocalizes with the expression of osteoblast-specific marker Col2.3emd (E, green), as indicated by the presence of yellow color (overlay image, F). To provide better orientation within the area of callus, same sections were stained by hematoxylin (G). Abbreviations: BM, bone marrow; CB, cortical bone.

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To track the population of SMA9+ cells into mature osteoblasts, we bred a bone-specific transgene into the SMA9 mice. The expression of SMA9+ cells (red-tdTomato) colocalized to Col2.3emd-expressing mature osteoblasts (green, SMA9/2.3GFP; Fig. 5D–5G). This finding is direct evidence for terminal differentiation of SMA9+ cells into mature osteoblasts in vivo. A representative image of a control fractured bone from an SMA9 mouse not injected with tamoxifen is shown in Figure 5C. No major leak of spontaneous Cre expression was detected following fracture of SMA9 untreated mice.

The majority of the cartilaginous callus cells were SMA9+ indicating differentiation into chondrocytes (Fig. 5B, 5F, 5G arrowheads). In addition to the morphological characterization of chondrocyte, we have introduced a chondrocyte lineage-directed reporter (Cyan) using a Col2a1 transgene. After 1 week of bone fracture in SMA9/Col2a1 mice, we have observed a population of dual SMA9/Col2a1Cyan-expressing cells indicating that the SMA9+ population labeled prior to fracture healing can gave rise to Col2a1-expressing cells (Supporting Information Fig. 5).

To define the contribution of the SMA9 mesenchymal progenitor cells to newly formed bone following completion of the fracture-healing process, we assessed the 6-week postfracture time point. Mice transgenic for SMA9/Col2.3emd were fractured and cell lineage was traced following callus remodeling (Fig. 6). The contribution of the SMA9+ cell was detected by persistence of the dual SMA9+/Col2.3emd-labeled cells (osteoblasts and osteocytes) within the new bone 6 weeks into the repair process (Fig. 6A–6D, arrows indicate numerous dual SMA9/Col2.3emd expressing osteoblasts and osteocytes).

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Figure 6. SMA9+ cells contribute to new bone formation following fracture healing. (A–C): Osteogenesis following fracture healing and callus remodeling is examined at 6 weeks following fractures. The expression of SMA9 (A), Col2.3emd (B), and the overlayed image was evaluated for the presence of the dual expressing mature osteoblast lineage cells (C). Hematoxylin staining of the same section has been completed following epifluorescence imaging (D). The expression of SMA9 is detected in the periosteal layer of cells (A, C, see arrowheads point to the expanded SMA9+ cells within periosteum), within the osteoblast layer (C, indicated by arrows), among osteocytes and within the muscle. High magnification (×20) clearly identifies the presence of numerous cells that express both SMA9 and Col2.3emd markers (C, yellow cells on the bone surface and within the bone matrix). For this experiment, unilateral fractures were performed in three mice. Abbreviations: BM, bone marrow; CB, cortical bone.

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DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSION
  8. Acknowledgements
  9. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  10. REFERENCES
  11. Supporting Information

Skeletal stem cell, as a term, has been proposed for the population of mesechymal progenitor cells residing within the bone marrow compartment [17, 18]. The most accepted markers to define human mesenchymal stem cells are STRO-1, recognizing a milieu of cell surface glycoproteins, and MCAM/CD146, a marker that has been associated with subendothelial perivascular cells [5, 19]. Recently, the expression of nestin-driven reporters has been associated with a population of bone marrow cells that can differentiate into mesenchymal lineages [20]. Although useful to isolate cells from bone marrow and primary cell cultures, these markers do not allow for lineage tracing of the progeny of the mesenchymal stem cell as they are only transiently expressed at certain stages of differentiation.

The origin of the mature osteoblast remains elusive. We hypothesize that the αSMA transgene expression could be a marker for a cell that can give rise not only to osteoblasts but also to other mesenchymal lineage cells. We have addressed this by using an αSMACreERT2 transgene aiming to confirm the progenitor potential of αSMA-expressing perivascular cell in vivo. We were able to trace the progeny of the perivascular cells into mature osteoblasts and osteocytes, the final stage of skeletal lineage differentiation. These data indicate that during the process of bone formation and remodeling, the αSMA transgene identifies skeletal stem cells.

These observations prompted us to define the surface markers in the cells identified by αSMAcherry as well as SMA9 expression. A high proportion of αSMAcherry+ and SMA9+ cells expressed markers associated with hematopoietic cells including CD45. The exclusiveness of CD45 as pan-hematopoietic marker has been recently challenged, indicating that mesenchymal progenitor cells can express markers currently used to define the hematopoietic lineage [21, 22].

Skeletal stem cell interactions with endothelial cells are critical for early invasion of vasculature during embryonic development and for the translocation of skeletal stem cells into the newly formed bone marrow cavity [23]. However, recent literature has proposed alternative definitions for the identity of the mesechymal progenitor cells regarding their expression of endothelial cell markers. Medici et al. [24] described cells with endothelial phenotype exhibiting the ability to differentiate into mesenchymal cell lineages. Other report, using a coculture model of human embryonic stem cells with mouse OP9 stromal cells, defined a common precursor for endothelial and mesenchymal cell lineages, termed mesenchymoangioblast [25]. These studies cannot be strictly compared as they are derived from different experimental models. However, they provide evidence for a close relationship between mesenchymal and endothelial lineage cells. In our study, we have observed the expression of αSMAcherry in proximity to endothelial cells, while very few or none of the αSMAcherry+ coexpressed CD31 marker. Nevertheless, when the progeny of the SMA9 cells was traced in vivo, we observed a weak coexpression of CD31 in approximately 5% of the (CD45/Ter119/CD11b), SMA9+ cells. The proportion of CD31+/SMA9+ cells represents approximately 30% of the cells positive for markers of hematopoietic lineage. These findings suggest the possibility that SMA9-labeled progenitor cells, located in the perivascular region, have the ability to mature into cells with an endothelial phenotype. In addition, within SMA9+ bone marrow population that are positive for hematopoietic markers, we observed approximately 20% of CD146+/SMA9+ cells, while there were very few or none CD146-expressing cells among periosteal layer or in vitro expanded SMA9+ cells. Future studies have to be aimed to define these populations using a combination of approaches and multiple markers for both mesenchymal and endothelial lineages.

To test functionally the proposed hypothesis, the osteogenic potential of SMA9-labeled cells was tested in a bone injury model. Our data indicate that the majority of the callus cells, including chondrocytes and osteoblasts, are derived from SMA9-expressing cells. Interestingly, the expansion of the SMA9 cells occurred both in periosteum and within bone marrow and resulted into SMA9+ cells with mature bone cell phenotypes contributing to the fracture-healing process.

CONCLUSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSION
  8. Acknowledgements
  9. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  10. REFERENCES
  11. Supporting Information

We have utilized the αSMACreERT2 transgenic approach to selectively identify and isolate progenitor cells that can differentiate into mesenchymal lineages in vitro and in vivo. In addition, we were able to identify mesenchymal progenitor cells that actively participate in the fracture healing process by differentiation into mature chondrocytes and osteoblasts. In combination with visual markers of mature lineages, this will allow for the identification of pathways important for lineage determination that could provide therapeutic targets for the enhancement of the regenerative processes.

Acknowledgements

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSION
  8. Acknowledgements
  9. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  10. REFERENCES
  11. Supporting Information

This work was supported by NIH/NIAMS AR059315-01 and AR055607-01 grants to I.K., NIH/NHLBI 1RC1HL100569-01 to H.L.A., and NIH/NIAMS AR043457 to D.W.R. S.P. and D.R. have been supported by Croatian Science Foundation.

REFERENCES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSION
  8. Acknowledgements
  9. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  10. REFERENCES
  11. Supporting Information

Supporting Information

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSION
  8. Acknowledgements
  9. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  10. REFERENCES
  11. Supporting Information

Additional Supporting Information may be found in the online version of this article.

FilenameFormatSizeDescription
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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