Exosomes derived from P2X7 receptor gene‐modified cells rescue inflammation‐compromised periodontal ligament stem cells from dysfunction

Abstract Although cellular therapy has been proposed for inflammation‐related disorders such as periodontitis for decades, clinical application has been unsuccessful. One explanation for these disappointing results is that the functions of stem cells are substantially compromised when they are transplanted into an inflammatory in vivo milieu. Considering the previous finding that P2X7 receptor (P2X7R) gene modification is able to reverse inflammation‐mediated impairment of periodontal ligament stem cells (PDLSCs), we further hypothesized that cells subjected to P2X7R gene transduction also exert influences on other cells within an in vivo milieu via an exosome‐mediated paracrine mechanism. To define the paracrine ability of P2X7R gene‐modified cells, P2X7R gene‐modified stem cell‐derived conditional medium (CM‐Ad‐P2X7) and exosomes (Exs‐Ad‐P2X7) were used to incubate PDLSCs. In an inflammatory osteogenic microenvironment, inflammation‐mediated changes in PDLSCs were substantially reduced, as shown by quantitative real‐time PCR (qRT‐PCR) analysis, Western blot analysis, alkaline phosphatase (ALP) staining/activity assays, and Alizarin red staining. In addition, the Agilent miRNA microarray system combined with qRT‐PCR analysis revealed that miR‐3679‐5p, miR‐6515‐5p, and miR‐6747‐5p were highly expressed in Exs‐Ad‐P2X7. Further functional tests and luciferase reporter assays revealed that miR‐3679‐5p and miR‐6747‐5p bound directly to the GREM‐1 protein, while miR‐6515‐5p bound to the GREM‐1 protein indirectly; these effects combined to rescue inflammation‐compromised PDLSCs from dysfunction. Thus, in addition to maintaining their robust functionality under inflammatory conditions, P2X7R gene‐modified stem cells may exert positive influences on their neighbors via a paracrine mechanism, pointing to a novel strategy for modifying the harsh local microenvironment to accommodate stem cells and promote improved tissue regeneration.


| INTRODUCTION
Periodontitis is a widespread disease characterized by inflammationinduced connective tissue insult, intrabony pocket formation, damage to the tooth-supporting apparatus, and eventual tooth loss (reviewed in Pihlstrom et al 1 ). For over a decade, the idea of stem cell-based periodontal therapy has brought hope for growing new tissues in periodontal pockets refractory to current regenerative paradigms, but the clinical translation of PDLSCs, the first cell source of choice for periodontal therapy, in patients suffering periodontitis has been disappointing to date (reviewed in Hu et al, 2 Novello et al, 3 and Nunez et al, 4 ; eg, see Reference 5). One reason for the lack of success in clinical practice is that PDLSCs are functionally compromised when they are placed in an inflammatory intrabony periodontal defect. 4,6 Due to incomplete knowledge of the underlying mechanism, 7-9 therapeutic approaches that protect PDLSCs from inflammation-induced dysfunction (eg, reduced stemness and aberrant differentiation) following transplantation are still in their infancy. 10,11 In our previous study, we identified the role of P2X7 receptor (P2X7R) in inflammation-compromised osteogenic differentiation of PDLSCs, and most importantly, we found that the cell impairment could be reversed by P2X7R gene modification. 8 This finding suggests that the transplantation of P2X7R gene-modified PDLSCs could lead to an improved therapeutic outcome. However, the large-scale use of gene-transferred cells in the clinic is associated with additional safety concerns. Further elucidation of the cellular and molecular events that occur when P2X7R gene-modified PDLSCs encounter an inflammatory microenvironment is key to advance the P2X7R-targeted strategy for cell-based tissue regeneration. 12 Growing evidence has demonstrated that exogenous transplanted cells may regulate and catalyze tissue regeneration via secretion of a spectrum of stimulatory factors as well as extracellular vesicles (EVs). [13][14][15] In this regard, these cells, instead of or in addition to directly participating in the growth of new tissue, act as initiators to trigger and orchestrate tissue regeneration based on an endogenous repair mechanism, including but not limited to mobilizing/activating resident cells and modulating the regenerative microenvironment. 16 In fact, the local inflammatory milieu not only impairs the therapeutic efficacy of transplanted cells but also negatively affects the regenerative potential of resident PDLSCs. 12 Hence, it needs to be determined whether cells subjected to P2X7R gene transduction can exert positive influences on other coexisting resident cells within an in vivo milieu. If this is true, transplantation of a small number of cells subjected to P2X7R gene modification would be able to modulate the local hostile microenvironment and act as an initiator to trigger tissue repair in future periodontal regenerative medicine applications.
To define the secretory influence of P2X7R gene-modified cells on other coexisting cells, the osteogenic potential of PDLSCs was investigated when they were placed in an inflammatory osteogenic microenvironment in the presence or absence of P2X7R gene-modified stem cell-derived conditional medium (CM-Ad-P2X7) and exosomes (Exs-Ad-P2X7). Furthermore, we explored the molecular changes in Exs-Ad-P2X7 and how the altered miRNAs were able to rescue inflammation-compromised PDLSCs from dysfunction. Our data may provide new information for the development of microenvironment-based strategies for tissue engineering and regeneration.

| Collection of cell conditioned media derived from various cultures
Human PDLSCs were isolated and modified to overexpress P2X7R by gene transfer methods (for additional details, see Appendix S1 in Supporting Information). Following culture for 24    Exosomes were extracted by using the Total Exosome Isolation Kit for Cell Culture Media (Invitrogen, Lithuania) from supernatants according to the manufacturer's protocol. In brief, the obtained supernatants were centrifuged at 2000g for 30 minutes and then transferred to sterile tubes. Following the addition of the reagent to the supernatants and incubation overnight at 4 C, the mixture was centrifuged at 4 C and 10 000g for 1 hour. When the supernatant of the mixture was aspirated, exosomes attached to the tubes were suspended in PBS and normalized by cell number (the cells they were isolated from) to ensure that 100 μL of phosphate buffer saline (PBS) suspension contained exosomes isolated from 10 6 cells. 17 Exosomes derived from P2X7R gene-modified PDLSCs were designated as Exs-Ad-P2X7R, while those derived from cells transfected with blank adenoviral vectors were designated as Exs-Ad-control.

| Group design
To test the effects of Exs-Ad-P2X7 on the osteogenic differentiation of PDLSCs under inflammatory conditions, exosomes were also isolated from PDLSCs transfected with blank adenoviral vectors (Exs-Ad-control) and untransfected PDLSCs (Exs-PDLSCs); PBS was used as the blank control for Exs (Exs-control). Finally, four groups (Exs-control, Exs-PDLSCs, Exs-Ad-P2X7, and Exs-Ad-control) were designed to investigate how the presence of Exs-Ad-P2X7 affects the osteogenic potential of PDLSCs.

| Cell osteogenic differentiation in response to exosome-based incubation
Exs suspension (100 μL) was added to 2 × 10 5 cells. 17 Based on various culture conditions (Exs-control, Exs-PDLSCs, Exs-Ad-P2X7, and Exs-Ad-control), the osteogenic differentiation of PDLSCs under inflammatory conditions was tested following the determined investigation time in terms of osteogenic differentiation-related gene/protein (BMP2, OCN, and Runx2) expression, ALP staining/activity analysis, and Alizarin red staining (Appendix S1 in Supporting Information).

| Identification of differentially expressed miRNAs in Exs-Ad-P2X7
Total RNA from exosomes was extracted with TRIzol Reagent (Invi- were used to analyze and process the data. After quantile normalization of the raw data, miRNAs for which at least three out of six samples had flags in the detection system were chosen for further data analysis. Differentially expressed miRNAs with statistical significance between the two groups were identified through Volcano plot filtering and fold change filtering. Hierarchical clustering was performed using R scripts. The active sequences of differentially expressed miRNAs are shown in Appendix S1 in Supporting Information. Then, quantitative real-time PCR (qRT-PCR) was used to validate the differentially expressed miRNAs as described in Appendix S1 in Supporting Information. Briefly, 1.5 μL of transfection reagent was mixed with 1.5 μL of 20 μM differentially expressed miRNA-mimic or miR-mimic NC and then incubated at room temperature for 10 minutes. The mixture was diluted in 300 μL of α-MEM and added to 24-well plates (cells at 40%-50% confluence). The medium was replaced with osteogenic culture medium 12 hours after transfection. Total RNA was extracted at day 2 or day 7 for further analysis. All transfection experiments were repeated in triplicate.

| Osteogenic differentiation of PDLSCs in response to miRNA-mimic transfection
The osteogenic differentiation of PDLSCs that were transfected with miR-mimic NC or different miRNA mimics related to differentially expressed miRNAs between Exs-Ad-P2X7 and Exs-Ad-control were tested by means of qRT-PCR (Appendix S1 in Supporting Information).
2.7 | Determination of target genes for differentially expressed miRNAs in Exs-Ad-P2X7

| Primary selection of target genes
The potential genes targeted by differentially expressed miRNAs between Exs-Ad-P2X7 and Exs-Ad-control were assessed using the online software available from PicTar, TargetScan, and mirBase. The genes that were targeted by at least two differentially expressed miRNAs and closely related to osteoblast differentiation and osteogenesis were chosen for further validation using qRT-PCR assays (for additional details, see Appendix S1 in Supporting Information).

| Validation of the selected genes
PDLSCs were seeded in six-well plates at a concentration of 1 × 10 5 cells/well, allowed to reach 80% confluence and then treated with Exs-control, Exs-PDLSCs, Exs-Ad-P2X7, and Exs-Ad-control for 7 days. The selected genes targeted by differentially expressed miRNAs in Exs-Ad-P2X7 was (were) validated by qRT-PCR and Western blot assays. Then, PDLSCs were transfected with miR-mimic NC or miRNA mimics of differentially expressed miRNAs, and the selected genes were tested by means of qRT-PCR and Western blot assays (for additional details, see Appendix S1 in Supporting Information).

| Luciferase reporter assay
The final validation of the relationship between target genes and differentially expressed miRNAs was performed by using the luciferase reporter assay as described in Li et al. 18 In brief, 293T cells were transfected with firefly luciferase transcript containing either a wildtype or mutant form of the selected genes and then transfected with differentially expressed miRNA-mimics; the luciferase reporter activity was assessed at 24 hours post-transfection. A dual-luciferase reporter assay system (Promega, Madison, Wisconsin) was used to measure the firefly and Renilla luciferase activities of the harvested cells. A luminometer (TD-20/20; Turner Designs, Sunnyvale, California) was used to quantify luciferase activities and to calculate the relative ratios.

| Elucidation of the functions of the target genes
The functions of the selected genes were finally elucidated using the associated siRNA or plasmid. In brief, PDLSCs were seeded in six-well plates at a concentration of 1 × 10 5 cells/well; after the cells reached 80% confluence in six-well dishes, siRNAs/plasmids for the target genes were transfected into PDLSCs using Micropoly-transfecter for cells according to the manufacturer's instructions. siRNA NC/plasmid NC (Likeli, Beijing, China) was used as the negative control. The osteogenesis of different transfected PDLSCs was detected by BMP2, OCN, and Runx2 gene expression, as described in Appendix S1 in Supporting Information.

| Statistical analysis
All results are presented as the mean and SD for at least n = 3; experiments for each cell line were performed independently and in triplicate. We used GraphPad Prism 7.03 software for statistical analysis.
Student's t test was used to analyze two unpaired groups, and oneway analysis of variance (ANOVA) followed by Tukey's post-test was used for multiple group analysis. Statistical significance was established at P < .05.

| Characterization of human PDLSCs
PDLSCs were isolated and characterized by their surface markers and multilineage potential, as reported previously. 8

| Exosomes derived from P2X7R genemodified stem cells (Exs-Ad-P2X7)
After 24 hours of culture in serum-free osteogenic culture media, Ad-P2X7-transfected PDLSC culture supernatants were collected to isolate cell-derived exosomes. When subjected to TEM observation, the obtained exosomes showed a cup-or sphere-shaped morphology with a diameter in the range of 100 to 160 nm ( Figure 2A). Nanosight analysis revealed that most isolated exosomes ranged in size from 120 to 163 nm ( Figure 2B). Western blot analysis of exosomes and PDLSC lysates showed that the exosomes from Ad-P2X7 transfected cells contained the exosomal markers Annexin V, CD9, Flotillin-1, EpCAM, and CD63 ( Figure 2C) but were negative for Alix and HSP70. The endocytosis of exosomes by PDLSCs is shown in Figure 2D. The isolated exosomes were labeled with PKH67 (green), and PDLSCs were incubated with the labeled exosomes at 37 C or 4 C for 4 hours.
PDLSCs incubated without the labeled exosomes at 37 C were used as a negative control. After 4 hours, the cells were immunostained with anti-tubulin antibody (red) and 4',6-diamidino-2-phenylindole (DAPI; blue) to verify whether the exosomes could be endocytosed by PDLSCs. The images showed that in the control group (negative control), PDLSCs were negative for the green fluorescent signal, while the green fluorescent signals were significantly stronger in the exosomeexposed group at 37 C; much lower green fluorescent signals were detected in the 4 C group.
Then, the different exosome suspensions were added to inflammatory osteogenic culture medium for further incubation. After 7 days of Data are presented as the mean ± SD for n = 4; *P < .05, **P < .01, and ***P < .001 indicate significant differences between the indicated columns. PDLSC, periodontal ligament stem cell incubation, BMP2 expression was threefold higher in the Exs-Ad-P2X7 group than in the Exs-control group (P < .001, Figure 3A). Runx2 and OCN expression was also higher in the Exs-Ad-P2X7 group than in the other groups ( Figure 3B,C). The Exs-PDLSCs group and Exs-Adcontrol group had similar BMP2, Runx2, and OCN expression levels, but all of these levels were higher than that of the Exs-control group ( Figure 3A-C). The protein expression was consistent with mRNA expression ( Figure 3D,E). The exosomes were also added to inflammatory osteogenic culture conditions, and PDLSCs were incubated for 7 or 21 days before the detection of ALP activity or Alizarin red staining. Both the ALP activity detection/staining and Alizarin red staining results suggested that the Exs-Ad-P2X7 group had better ALP activity and mineralized nodule formation than the Exs-PDLSCs group or Exs-Ad-control group (P < .05, .01, or .001, Figure 3F,G). The ALP activity and mineralized nodule formation in the Exs-control group were the lowest. Further qRT-PCR analysis revealed that miR-3679-5p, miR-6515-5p, F I G U R E 3 Incubation of PDLSCs in cultures containing Exs-Ad-P2X7 promoted osteogenic differentiation of PDLSCs in an inflammatory microenvironment. A, BMP2, B, OCN, and, C, Runx2 gene expression in PDLSCs (qRT-PCR assay) following a 7-day incubation in cultures containing Exs-control, Exs-PDLSCs, Exs-Ad-P2X7, and Exs-Ad-control. D, BMP2, OCN, and Runx2 protein expression and, E, quantitative analysis in PDLSCs (Western blot analysis) following a 7-day incubation in cultures containing Exs-control, Exs-PDLSCs, Exs-Ad-P2X7, and Exs-Ad-control. F, ALP staining and quantitative analysis of ALP activity in PDLSCs following a 7-day incubation in cultures containing Exs-control, Exs-PDLSCs, Exs-Ad-P2X7, and Exs-Ad-control. G, Alizarin red staining and quantitative analysis of the mineralized nodes formed by PDLSCs following a 21-day incubation in cultures containing Exs-control, Exs-PDLSCs, Exs-Ad-P2X7, and Exs-Ad-control. Data are presented as the mean ± SD for n = 4; *P < .05, **P < .01, and ***P < .001 indicate significant differences between the indicated columns. PDLSC, periodontal ligament stem cell and miR-6747-5p were significantly increased in Exs-Ad-P2X7 compared with Exs-Ad-control ( Figure 4D), in accordance with the sequencing results. The other three miRNAs were not consistent with the sequencing results. Thus, we used miR-3679-5p, miR-6515-5p, and miR-6747-5p as our study genes in subsequent experiments.
OCN and Runx2 all increased over 1.2-fold in each miR-mimic group.
However, the protein expression increase observed for Runx2 was not as obvious as the mRNA expression result, as the increase in Runx2 expression in all groups failed to reach statistical significance ( Figure 5D,E). Both BMP2 and OCN protein expression increased over twofold in all miR-mimic groups compared with the miR-mimic NC group.

| miR-3679-5p and miR-6747-5p binding to the GREM-1 protein contributed to the cellular functions of Exs-Ad-P2X7
Prompted by these results, we then sought to investigate the molecular mechanism underlying Exs-Ad-P2X7-enhanced osteogenic differentiation of PDLSCs in inflammatory microenvironments.
Potential gene targets of miR-3679-5p, miR-6515-5p, and miR-6747-5p were assessed using the online software available from Pic-Tar, TargetScan, and mirBase. More than 2000 genes were predicted to be potential targets of differentially expressed miRNAs. We focused on molecules that were closely related to osteogenic differentiation, and a survey was also performed using the PubMed website to find this miRNA function in other areas of study. Finally, 37 genes were selected and investigated by a qRT-PCR assay ( Figure 6A). The results suggested that some molecules, such as HOXA2, CHRD, OSX, LIFR, and EGFR, were significantly increased in Exs-Ad-P2X7-treated PDLSCs in inflammatory osteogenic microenvironments, and among all the increased molecules, the transforming growth factor beta (TGF-β)/bone morphogenetic protein (BMP) signaling family showed a similar increase in the Ad-P2X7R-Exs-treated group. Thus, we hypothesized that the TGF-β/BMP signaling pathway plays a central role in exosome-enhanced osteogenic differentiation of PDLSCs in inflammatory microenvironments. GREM-1, a BMP antagonist from the DAN family, plays a key role in regulating the TGF-β/BMP signaling pathway. It can bind to BMPs and prevent BMPs from interacting with BMP receptors (provided by NCBI Reference Sequences; https:// www.ncbi.nlm.nih.gov/gene/26585). Because GREM-1 decreased significantly in Exs-Ad-P2X7-treated PDLSCs ( Figure 6B,C) and because transduction of miR-3679-mimic, miR-6515-mimic, and miR-6747mimic into PDLSCs could also decrease GREM-1 expression ( Figure   6D,E), we hypothesized that GREM-1 may be involved in miR-3679-, miR-6515-, and miR-6747-mediated changes in PDLSCs.
Furthermore, we used a luciferase reporter assay system to confirm the relationship between GREM-1 and differentially expressed miR-3679-5p, miR-6515-5p, and miR-6747-5p. The results showed that the relative luciferase activity was reduced by 28% in the miR-3679-mimic group (P < .01; Figure 6F) and 22% in the miR-6747mimic group compared with the miR-3679-mimic group (P < .01; Figure 6H), while transduction of the miR-6515-mimic did not change the relative luciferase activity compared with that of the NC-mimic group ( Figure 6G). This result suggests that GREM-1 is the target gene for miR-3679-5p and miR-6747-5p but not for miR-6515-5p.

| DISCUSSION
Multiple studies have proven that the inflammatory microenvironment in defective periodontal sites hinders stem cell-based periodontal tissue regeneration. 4,6 Further elucidation of the underlying mechanism that occurs when stem cells encounter an inflammatory microenvironment will definitely benefit stem cell-based regeneration therapy. The P2X7 receptor (P2X7R) belongs to the adrenergic receptor family 19 and plays important physiological and pathological roles in vivo, [20][21][22] including in periodontitis. 23,24 Our previous study proved that P2X7R gene modification could significantly enhance the osteogenesis of PDLSCs through the PI3K-Akt-mTOR signaling pathway. 8 In fact, stem cells could improve the local microenvironment of the defect site not only by their own multiplication and differentiation but also by mobilizing the endogenous regeneration ability of resident cells though the molecules they secrete. 25,26 These transplanted stem cells can secrete a spectrum of stimulatory factors as well as EVs [13][14][15] to enhance tissue regeneration. Coincidentally, the available literature supports the notion that P2X7R could enhance cell paracrine ability by enhancing cell membrane blebbing, 27 cytokine release, 28,29 and exosome production in multiple cell types. [30][31][32] To explore the potential modulatory effect of P2X7R gene-modified PDLSCs on their neighbors when they coexist, conditioned medium from P2X7R gene- improved by genetic manipulation, as reported by several studies, [44][45][46] because they faithfully reflect the genomic characteristics of their parent cells and thus have functions similar to those of their parent cells. 37,47 In this study, we isolated exosomes (Exs-Ad-P2X7) from CM-Ad- reported that P2X7R could regulate bone marrow mesenchymal stem cells (BMMSC) and bone homeostasis through Tet1-and Tet2-controlled P2X7R demethylation and exosome release.
The lipid bilayers of exosomes can effectively protect multiple RNAs from degradation. One previous study reported that most miRNAs in serum were present in exosomes. 49 MicroRNA (miRNA) is a class of posttranscriptional regulator and one of the most important cargoes in exosomes. 50 It is currently well recognized that miRNAs can bind to 60% of all genes and repress target gene expression, 51,52 regulating multiple physiological and pathological processes, including the osteogenic differentiation process. [53][54][55] In this study, we sequenced the miRNA profiles of Exs-Ad-P2X7 and Exs-Ad-control from three different individuals and found that miR-3679-5p, miR-6515-5p, and miR-6747-5p increased significantly in Exs-Ad-P2X7 ( Figure 4). Further study demonstrated that miR-3679-5p, miR-6515-5p, and miR-6747-5p played positive roles in the osteogenic differentiation of PDLSCs in the inflammatory microenvironment ( Figure 5). A previous study reported that miR-302/367 could maintain the pluripotency of human embryonic stem cells and regulate their differentiation. 56 Liu et al 57 reported that the expression of miR-3679-5p was significantly increased in plasma from coronary artery calcification patients in an independent clinical matched cohort. Another study reported that miR-3679 and miR-4274 were associated with bone mineral density in the osteoporotic phenotype. 58 59 Hence, specific target genes of differentially expressed miRNAs in Exs-Ad-P2X7 were predicted by PicTar, TargetScan, and mirBase in the present study. Focusing on target genes of three differentially expressed miRNAs that were closely related to osteoblast differentiation and osteogenesis, we found that the TGF-β/BMP signaling family was increased in the Ad-P2X7R-Exs-treated group, suggesting that the TGF-β/BMP signaling pathway plays a central role in exosome-enhanced osteogenic differentiation of PDLSCs in inflammatory microenvironments. Because most miRNAs regulate cell function by posttranscriptional inhibition of gene expression, we then focused on investigating genes that were downregulated in the TGFβ/BMP signaling family. We finally identified that GREM-1 was most likely a common target gene of miRNA-3679, miR-6515, and miR-6747. GREM-1 is a BMP antagonist belonging to the DAN family.
GREM-1 binds to BMP2, BMP4, and BMP7 and inhibits BMP signaling. 60 Further validation assays using a luciferase reporter assay confirmed that mechanistically, GREM-1 was the direct target gene of the differentially expressed miRNAs miR-3679-5p and miR-6747-5p (Figure 6). A functional test of GREM-1 suggested that there was an indirect connection between GREM-1 and miR-6515. Inhibition or induction of GREM-1 changed the osteogenic differentiation of PDLSCs under inflammatory conditions (Figure 7). Similar results have been reported by Ghuman, showing that GREM-1 can also limit coronal alveolar bone regenerative potential during oral and periodontal surgery by inhibiting BMP-induced bone formation. 61 Other studies have shown that GREM-1 plays an important regularly role in organogenesis of the limbs and kidneys, 62 and mutations in GREM-1 lead to abnormalities of the limbs, lungs, and kidneys in mice. 63 Taken together, these results show that P2X7R plays an important role in regulating and catalyzing tissue regeneration under inflammatory culture conditions. Cells subjected to P2X7R-gene modification can exert a positive influence on other coexisting resident cells though the exosomes (Exs-Ad-P2X7) they secrete. Differentially expressed miRNAs in exosomes and their target gene GREM-1 are involved in this process, as summarized in Figure S4. The utilization of Exs-Ad-P2X7 in the regeneration process instead of direct transplantation of adenovirus-transfected stem cells into defective sites not only improves the safety and effectiveness of stem cell therapy without evoking additional side effects, but also provides a feasible and promising method for the clinical treatment of periodontitis. The limitations of the present study should also be acknowledged because the direct target gene of miR-6515-5p has not been identified, and more in vivo experiments are needed to verify the positive influence of P2X7R on tissue regeneration. However, with stringent proof-of-concept strategies, it might be possible to translate P2X7R gene modification from rodents to human patients.

| CONCLUSION
In this study, we found that in addition to reversing inflammationmediated impairment in cells subjected to gene transduction, the transduced cells also exert positive influences on other coexisting cells via an exosome-mediated paracrine mechanism. Our data suggest that either transplantation of a small number of P2X7R-gene-modified cells or the use of their exosomes could serve as an initiator to modify the local inflammatrory microenvironment to accommodate stem cells, either exogenously transplanted or endogenously mobilized, and lead to improved tissue regeneration.