The Role of Mesenchymal Stromal Cells in Tissue Repair and Fibrosis

Abstract

Significance:

The present review covers an overview of the current understanding of biology of mesenchymal stromal cells (MSCs) and suggests an important role of their differential potential for clinical approaches associated with tissue repair and fibrosis.

Recent Advances:

Genetic lineage tracing technology has enabled the delineation of cellular hierarchies and examination of MSC cellular origins and myofibroblast sources. This technique has led to the characterization of perivascular MSC populations and suggests that pericytes might provide a local source of tissue-specific MSCs, which can differentiate into tissue-specific cells for tissue repair and fibrosis. Autologous adipose tissue MSCs led to the advance in tissue engineering for regeneration of damaged tissues.

Critical Issues:

Recent investigation has revealed that perivascular MSCs might be the origin of myofibroblasts during fibrosis development, and perivascular MSCs might be the major source of myofibroblasts in fibrogenic disease. Adipose tissue MSCs combined with cytokines and biomaterials are available in the treatment of soft tissue defect and skin wound healing.

Future Directions:

Further investigation of the roles of perivascular MSCs may enable new approaches in the treatment of fibrogenic disease; moreover, perivascular MSCs might have potential as an antifibrotic target for fibrogenic disease. Autologous adipose tissue MSCs combined with cytokines and biomaterials will be an alternative method for the treatment of soft tissue defect and skin wound healing.

Yoshikiyo Akasaka, MD, PhD

Scope and Significance

Mesenchymal stromal cells (MSCs) have broad differentiation plasticity and their differentiation potential are dependent on their tissue of origin. They have been identified by in vitro characteristics based on their differentiation potential while their biological properties and in vivo identities are less understood.

This review covers an overview of the current understanding of biology of MSCs and of the characterization of MSCs with their differential potential associated with tissue fibrosis. Recent advances in regenerative plastic surgery using adipose tissue MSCs is also discussed.

Translational Relevance

Genetic fate tracing technique has enabled the delineation of cellular hierarchies and examination of MSC cellular origins. This technique has led to the characterization of perivascular MSC populations that may contain perivascular mesenchymal progenitor cells (MPCs) capable of differentiating to specific tissue cells. Autologous adipose tissue MSCs combined with cytokines and biomaterials led to the advance in tissue engineering for regeneration of damaged tissues.

Clinical Relevance

Recent investigation showed that perivascular MSCs might be the origin of myofibroblasts during fibrosis development, and pericytes might be the major source of myofibroblasts in fibrogenic disease. Clarification of the roles of perivascular cells may enable new approaches in the treatment of fibrogenic disease; moreover, perivascular MSCs might have potential as an antifibrotic target for tissue fibrosis. Autologous adipose tissue MSCs combined with cytokines and biomaterials will be an alternative method for the treatment of soft tissue defect and skin wound healing.

Background

Mesenchymal stem cells or MSCs?

Stem cells were first discovered in the hematopoietic system by Till and McCulloch, who showed that bone marrow (BM) cells were able to produce multilineage mature hematopoietic cells while retaining the ability to self-renewal.¹ The colony-forming units in the spleen (CFU-S) assay established by Till and McCulloch has been used to identify primitive BM cells based on the forming colonies of mature hematopoietic cells in vivo.¹

The same tissue was subsequently examined by Friedenstein et al. who described that adherent and nonhematopoietic cells in BM were able to form fibroblastoid cells in vitro.² Since BM-derived fibroblastoid cells are heterogenous, limited dilution of the heterogenous BM cells allowed the isolation of individual cell clones, and homogeneous cell populations obtained from BM cells are termed colony-forming unit-fibroblasts (CFU-F).^2
–4

In the 1970s, Dexter et al. reported that BM fibroblastoid cells could sustain the growth of hematopoietic stem cells without supplementation of growth factors.^5

–8 In this system, the culture achieved homeostasis with the self-renewal of progenitor cells, indicating that BM fibroblastoid cells have an enhanced ability to sustain the growth of hematopoietic cells through secretion of growth factors.

The autoradiography tracing technique allowed us to identify BM cells that proliferate in the inner periosteal surface of human BM, which were termed preosteoblasts. The technique demonstrated that preosteoblasts were able to differentiate into osteogenic, adipogenic, chondrogenic, and myogenic mesenchymal cells in vitro,⁹ and the cells were termed “mesenchymal stem cells.”¹⁰ These findings were confirmed by Owen and Friedenstein.³ Further studies showed that mesenchymal stem cells were capable of differentiating into endothelial cells, cardiomyocytes, hepatocytes, and neural cells.^11

–14 However, in light of evidence that fibroblastoid cells do not always exhibit self-renewal and the ability to differentiate into multiple lineages, the term “mesenchymal stem cells” has been recognized as incorrect.^15,16

In response to the disagreement, the International Society for Cell and Gene Therapy (ISCT) recommended that these cells be commonly known as Mesenchymal Stromal Cells and that the widely recognized acronym (MSC) be used.¹⁷ The ISCT also developed basic criteria for defining human MSCs: MSCs must be plastic adherent and exhibit trilineage differentiation into adipocytes, chondrocytes, and osteocytes.¹⁸ The criteria additionally required the expression of CD105, CD73, and CD90 in MSCs, but not the expression of CD45, CD34, CD14, or CD11b, and CD79α or CD19. CD271 and CD146 were also identified as MSC markers.

The expression of CD271 and CD146 was detected in uncultured MSCs; however, downregulated expression of both molecules was observed under culture conditions.^19
–21 The criteria for MSCs in mice are that they express CD106 and stem cell antigen-1 (Sca-1), but lack expression of CD45, CD11b, and CD31.²² Uder et al. summarized a list of MSC markers and compared their expression patterns between humans and other mammals.²³ They showed that CD29 and CD44 were expressed on MSCs in all species, whereas only CD166 was expressed in human,²⁴ rats,²⁵ and sheep.²⁶ In addition, CD166 was expressed on osteoprogenitor cells, but not on differentiated osteocytes.

MSCs can be isolated from several tissues other than BM, such as adipose,²⁷ placenta,²⁸ skin,²⁹ umbilical cord blood,³⁰ umbilical cord perivascular cells,³¹ umbilical cord Wharton's jelly,³² dental pulp,³³ amniotic fluid,³⁴ and synovial membrane.³⁴ Although the ISCT guidelines provide the common properties of MSCs,³⁵ MSCs derived from different tissues exhibit marked differences in their rates of differentiation. The perivascular niche has been proposed as the source of MSCs in various tissues.³⁶ Direct analysis of presumptive perivascular MSCs has revealed that these cells are functionally diverse.³⁷ Differences between populations of MSCs from different sources are also observed under natural conditions.

Some studies have recommended that the term MSC be replaced with postnatal stem cells or tissue-specific stem cells, which is reflected in the recent ISCT recommendations.³⁸ Global standardized methods for the isolation, expansion, and identification of MSCs have not been established. Therefore, the multipotential capabilities of MSCs require mechanistic elucidation both in vivo and in vitro.¹⁵ Disputes regarding the nomenclature, definition, and characterization of MSCs were enumerated by Keating³⁹ as follows: (1)

The term “mesenchymal stem cell” should be used to specifically describe a cell with self-renewal and differentiation characteristics.

(2)

MSCs should be categorized as cultured or primary. This is an important distinction because the characteristics are likely to differ and would avoid confusion when comparisons are made between studies.

(3)

The source of MSCs should be specified (e.g., adipose, BM, cord blood, etc.).

(4)

Species should be identified. This information is not always stated in the text of publications and has led to confusion in the past.

(5)

Minimum criteria for the surface marker profile need to be revisited and are likely to vary among species.

This new approach appears to generate confusion, by referring to the literature of Jiang and Scharffetter-Kochanek who showed self-renewal and pluripotent differentiation capability in MSCs.⁴⁰ This new approach to the definition of MSCs may help to inform the investigation of MSCs rather than to serve as a classification.³⁹

Cell markers for MSCs

MSCs have been identified by a combination of positive and negative makers; MSCs must be positive for CD73, CD90, and CD105, and negative for CD45, CD34, CD14 or CD11b, CD79α or CD19, and HLA-DR. CD271⁺ BM human cells in combination with positive status of Thy-1 (CD90) and VCAM-1 (CD106) were enriched for an MSC population in vitro.¹⁹ CD146 and CD105⁴¹ or IntegrinαV and platelet-derived growth factor receptor (PDGFR) α were also found in human BM cells enriched for MSCs.⁴² In addition, STRO-1, stage-specific embryonic antigen (SSEA)-1, CD73, and CD49a have been used to isolate human bone marrow-derived MSCs (BM-MSCs).^43

–47

Matsuzaki and colleagues discovered novel markers in murine MSCs. They found that PDGFRα⁺/Sca-1⁺ murine BM cells are enriched for CFU-S and that this population met the criteria of trilineage differentiation in vitro.^48,49

The expression of MSC markers differs between in vivo and in vitro. Uncultured MSCs are positive for CD271 and CD146, whereas both molecules show changes in expression when the cells are cultured in vitro.^19
–21 CD271 expression is downregulated in MSCs in vitro. CD146 expression is upregulated in MSCs when isolated in a culture system, whereas hypoxic conditions in MSCs downregulate CD146 expression in vitro.²¹ Thus, specific culture conditions can reveal the characteristics of MSCs. In vitro features of MSCs are not always indicative of in vivo function. It should be noted that the variability of in vitro characteristics of MSCs can be broader compared with their cells of origin.

MSCs are localized to the perivascular region, but the specific markers of perivascular MSCs have not been fully identified. Zhao et al. reported glioma-associated oncogene homolog 1 (Gli1) as a marker of perivascular MSC-like cells in mouse incisor.⁵⁰ Genetic labeling techniques for identification of marker localization in mice revealed that BM Gli1⁺ MSC-like cells reside along the BM sinusoids and arterioles, possessing the characteristics of pericyte-like MPC or endosteal MSCs.⁵¹

On the other hand, leptin receptor (LepR) is expressed in murine BM perivascular cells and LepR⁺ cells were found in a population of CD51⁺/PDGFRα⁺ cells corresponding to multipotent MSCs.⁵² Genetic labeling techniques revealed that BM LepR⁺ MSCs reside in the area adjacent to the endosteum, possessing the characteristics of pericyte-like MPC. Differences in the distribution of Gli1⁺ and LepR⁺ cells in murine BM were clarified by Schneider et al. who showed no expression of LepR in Gli1⁺ cells and that the two types of perivascular MSC markers might be differentially expressed in murine BM.⁵¹ These findings clearly indicate the heterogeneity in the distribution of MSC markers in murine BM.⁵³

Immune modulation by MSCs

In vitro studies showed that MSCs can differentiate into epidermal cells, keratinocytes, and endothelial cells.⁵⁴ When transplanted into wounds, MSCs promote the proliferation of fibroblasts and activate fibroblast-to-myofibroblast transition. Sasaki et al. demonstrated that BM-MSCs contributed to wound repair through their differentiation into keratinocytes, endothelial cells, and pericytes.⁵⁵ However, due to the low rate of engraftment of exogenous MSCs in wounds, MSCs may contribute to the formation of granulation tissues through their immunomodulatory functions through the secretion of cytokines, chemokines, and proangiogenic factors.⁵⁶

MSCs have been shown to attenuate fibrosis by inhibiting wound contraction⁵⁷ and decreasing the number of myofibroblasts in the later phase of healing.⁵⁸ BM-tumor necrosis factor-stimulated gene-6 (TSG-6), which is released from MSCs, plays an important role in inhibiting the formation of granulation tissue formation in the later phase of healing.

The anti-inflammatory role of MSCs is their most prominent beneficial effect on wound healing. In acute wounds, MSCs inhibit the tumor necrosis factor (TNF)-α-dependent inflammation and release TSG-6,⁵⁹ thereby increasing the number of anti- inflammatory M2 macrophages.⁶⁰ Human MSCs attenuate the secretion of proinflammatory cytokines, such as INF-γ,⁶¹ interleukin (IL)-12,⁶² and TNF-α.⁶¹ MSCs also upregulate the expression of anti-inflammatory cytokines, including IL-10.^61,63
–65 Human BM-MSCs inhibit the proliferation of CD4⁺ and CD8⁺ T cells.

MSCs have been shown to suppress the proliferation of B cells and NK cells. Therefore, MSCs play an important role in attenuating the acute immune response in the early phase of healing. In the presence of low levels of TNF-α and INF-γ, MSCs promote inflammatory responses, implying the importance of the inflammatory environment for the immune regulation of MSCs.¹⁴ Ha et al. recently reported a role for MSC-derived exosomes in anti-inflammatory responses.⁶⁶

Advances in MSC therapy

The stromal vascular fraction (SVF) in adipose tissues contains adipose-derived stem cells (ASCs) that proliferate and differentiate into skin cells and also promote the regeneration of damaged cells. In this process, ASCs exhibit the ability to migrate and differentiate into dermal fibroblasts, endothelial cells, and keratinocytes without losing the ability for differentiation during transplantation.^67
–69 The ability of different biomaterials combined with ASCs to support the growth of recipient cells has been investigated. Scaffolds combined with ASCs may be effective in the treatment of bone and cartilage injuries, scars, and burn injuries.

In the treatment of soft tissue defects, platelet-rich plasma (PRP) and hyaluronic acid (HA) dressings effectively promoted the re-epithelialization of wounds with post-traumatic bone exposure^70,71 as well as complete wound healing in patients with hidradenitis suppurativa.⁷² Based on this background, Gentile et al. showed that SVF-enhanced autologous fat grafts combined with PRP were an effective treatment for the correction of scars on the face.⁷³

Scioli et al. reported that a collagen type I scaffold culture combined with PRP and insulin was applicable for the treatment of osteochondral defects based on enhancements in the chondrogenic/osteogenic differentiation of ASCs.⁷⁴ Furthermore, autologous ASCs promoted the regeneration of widespread traumatic calvarial bone defects and were effective in the treatment of tracheomediastinal fistulas.^75,76 The combined use of PRP and SFV/ASCs in fat grafting was also found to be effective for facial rejuvenation,⁷⁷ scars on the face,⁷³ and soft tissue defects and breast reconstruction.⁷⁸ In addition, an intra-articular injection of ASCs attenuated painful osteoarthritis by promoting the regeneration of cartilage.⁷⁹

Hair follicles contain MSCs with the ability for continuous self-renewal, differentiation, and supporting hair growth. Human MSCs derived from hair follicles have attracted attention because of their differentiation potential and accessibility.⁸⁰ Gentile et al. demonstrated that PRP and micrografts, including autologous human follicle mesenchymal stem cells promoted hair regrowth in androgenetic alopecia.⁸¹ They also showed that micrografts, including human intra- and extradermal adipose tissue-derived hair follicle stem cells were useful for the treatment of hair loss in androgenetic alopecia.⁸²

The same investigators successfully isolated autologous human activated PRP (AA-PRP) and demonstrated the efficacy of AA-PRP in the treatment of hair growth and wound healing, with the therapeutic effects satisfying with consensus quality criteria.⁸³

Gentile and Garcovich reviewed the efficacy of PRP, adipose-derived mesenchymal stem cells (AD-MSCs), and biomaterials as therapeutic approaches for chronic skin wounds, soft tissue defects, and skin defects.⁸⁴ The findings obtained indicated the safety and efficacy of the human allogeneic use of ASCs.⁸⁵

Gentile et al. also demonstrated the safety and utility of fat grafts with ASCs for breast reconstruction and engineered fat grafts enhanced with adipose-derived SVF cells.^86,87 Furthermore, they suggested the potential of human ASCs in breast reconstruction and the effectiveness of adjustments to processing quantitative standards for the isolation of AD-MSCs.^88,89 Gentile et al. discovered supercharged-modified nanofat grafting as a novel fat grafting technique.⁹⁰ In addition, Cervelli et al. assessed the platelet-rich lipotransfer method as an alternative strategy for treating soft tissue defects.⁹¹

Regarding the effectiveness of human MSCs against disease, Yaochite et al. showed that the administration of MSCs derived from type 1 diabetes mellitus patients into diabetic mice reversed hyperglycemia and ameliorated hyperglycemia.⁹² Alves et al. reported that the single administration of human ASCs into mice with colitis protected against acute intestinal inflammation and improved the clinical disease score.⁹³

Discussion of Findings and Relevant Literature

MSC cellular origins: perivascular MSCs

The accumulated evidence indicates that MSCs are associated with the vasculature. Hoshino et al. demonstrated that BM adventitial cells expressed MSC markers, that is, they possessed the ability to differentiate into adipocytes, osteoblasts, and smooth muscle cells in vitro.⁹⁴ Subsequent studies demonstrated that BM-MSCs were highly enriched in a CD271⁺ cell population.¹⁹ Tormin et al. found that perivascular CD271 expression was confined to tunica adventitial cells,²¹ indicating that BM adventitial cells could possess characteristics of MSCs.

Further investigation revealed that pericytes express MSC markers, such as 3G5, neural/glial antigen 2 (NG2), Sca-1, STRO-1, Vimentin, α smooth muscle actin (αSMA), Thy-1, V-CAM1, and PDGFRβ.⁹⁵ Crisan et al. reported that vascular pericytes expressed CD146, NG2, PDGFRβ, and MSC markers (CD44, CD73, CD90, and CD105) in human skeletal muscle, the pancreas, the adipose tissues, and the placenta. Pericytes isolated from skeletal muscle or nonmuscle tissues expressed MSC markers in vitro and exhibited osteogenic, chondrogenic, and adipogenic potentials at the clonal levels.^96,97

It has been suggested that crosstalk between endothelial cells and MSCs plays a regulatory role in MSCs in the perivascular niche. However, the specific extracellular mediators that regulate growth and differentiation of MSCs are not fully known.⁹⁸ El Agha et al. showed that a mesenchymal cell is a nonepithelial cell that often shows a stellate morphology, while a resident mesenchymal cell is a mesenchymal cell residing in each organ, but not originating from the circulatory system.⁹⁹

Since organ-resident perivascular MSCs displayed common features with BM-MSCs regarding cellular marker expression in vitro, organ-resident perivascular MSCs are termed “MSC-like cells.”³⁹ BM-MSCs and MSC-like cells appear to directly contribute to myofibroblast formation in fibrosis development.

Previous studies have demonstrated that isolated cells positive for pericyte markers are enriched with MSCs. Pericytes are known to participate in the formation, remodeling, and maintenance of the vascular system through intimate interactions with endothelial cells.^100,101 The phenotype of pericytes varies based on the resident tissue and vessel size.¹⁰² Pericytes in the liver and kidney are called as hepatic stellate cells and glomerular mesangial cells, respectively.^103,104

The density of pericytes varies between different organs, and the proportion of the endothelial abluminal surface that is covered by pericytes also varies. The vasculature in the central nervous system is considered to be covered by pericytes, with a 1:1–3:1 ratio between endothelial cells and pericytes.^105,106 This ratio was reported to be markedly lower in human skeletal muscle, with an endothelial-to-pericyte ratio of 100:1.^95,107 The central nervous system is considered to have the highest rate of coverage at about 30%.^105,106 PDGFRβ, CD13, αSMA, NG2, regulator of G-protein signaling 5 (RGS5), and 3G5 have been identified as pericyte markers.^{108

–116}

Targeted deletion of the retention motif in PDGFRβ resulted in hypoplasia and partial detachment of pericytes in mice,¹¹⁷ indicating the role of PDGF-B/PDGFRβ signaling as a matrix-bound factor. Recent analyses revealed new pericyte markers such as Annexin A5 (Anxa5) and the homeodomain transcription factor Msx1.^118,119 No single pericyte-specific marker has been found and the use of a combination of several different markers is required. Moreover, an obvious difference in the expression of NG2/αSMA was observed between organs in humans; NG2⁺ pericytes are found in arterioles and capillaries, but not in venules.⁹⁶ Pericytes in capillaries are NG2⁺/αSMA⁻, venules are NG2⁻/αSMA⁺, and arterioles are NG2⁺/αSMA⁺, and CD146 and PDGFRβ are expressed in every type of pericyte.¹²⁰

Hannan et al. reported a useful method for the detection of pericytes strongly labeled with tdTomato in lung fibrosis in Myh11-CreERT2 ROSA STOPfl/fl tdTomato reporter mice. Therefore, by using the tdTomato marker in Myh11-CreERT2 ROSA STOPfl/fl tdTomato reporter mice, it may be possible to define tdTomato-positive cells as pericytes in fibrotic tissues.¹²¹

Previous evidence indicates that MSCs might be derived from pericytes.^97,122 Pericytes have been shown to differentiate into osteoblasts, chondrocytes, and adipocytes. Other types of mesenchymal cells shown to differentiate from pericytes include fibroblasts, neurons, glia cells, smooth muscle cells, and myogenic cells.^123
–125 However, Sasaki et al. demonstrated that marrow-derived MSCs differentiated into endothelial cells, keratinocytes, and pericytes.⁵⁵ Since pericytes fulfil the definition criteria for MSCs and show differentiation potential like MSCs, the question remains whether MSCs are identical to pericytes.^126,127 This ambiguity is because the identification of pericytes is based on a small number of pericyte markers, such as NG2 and PDGFRβ.^128,129

Furthermore, there is difficulty in determining whether MSCs and pericytes represent counterparts of the same cell population in vivo, due to the heterogeneity of MSC and pericyte immunophenotypes observed in many organs. CD146 is one of the defining markers of pericytes. Based on the finding that BM-derived CD146⁺ MSCs have been shown to retain more stem cell-like potential, it is possible that CD146⁺ pericytes are precursors to MSCs.¹³⁰

Supporting evidence was reported by Bouacida et al. who showed that pericyte-like cells exhibited enhanced stemness, with higher expression of the stem cell markers, octamer-binding transcription factor 4 (OCT4) and sex-determining region Y-box 2 (SOX2), and markedly lower levels of protein expression associated with osteocyte, chondrocyte, and adipocyte differentiation.¹³¹ Another study supported the notion of pericyte stem cell potential with enhanced ability to proliferate and migrate in response to hypoxic stress and injury in multiple tissues.⁹⁸

Corselli et al. provided evidence that human adipose-derived MSCs are adventitial cells within the vascular wall.¹³² They showed that CD34⁺/CD146⁻/CD31⁻ adventitial cells from adipose tissue expressed MSC markers, such as CD44, CD73, CD90, and CD105, in situ. These cells were able to differentiate into adipogenic, chondrogenic, and osteogenic cells in vitro. Moreover, cultured adventitial cells did not express any pericyte markers. However, adventitial cells cultured with angiopoetin-2 showed upregulated pericyte markers. Therefore, MSCs might possess specific characteristics of adventitial cells and precursors of pericytes.

Recently, Zhao et al. reported on the relationship between MSCs and pericytes in mouse incisor, and they showed that Gli1⁺ cells are the most primitive MSCs with a high potential to be the precursor of all stromal cells following injury. These Gli1⁺ cells are perivascular adventitial cells and did not express typical MSC markers in vivo. In contrast, NG2⁺ pericytes expressed the classical MSC markers, but did not originate other stomal cells.⁵⁰ Cre-recombinase analysis demonstrated that NG2⁺ pericytes originated from Gli1⁺ cells in vivo, whereas the same cells did not express MSC markers in vivo. In contrast, cultured Gli1⁺ and NG2⁺ cells expressed typical MSC markers, showing a greater ability to differentiate into adipogenic, osteogenic, and chondrogenic cells in vitro.

When MSCs were cultured, 95% were derived from Gli1⁺ cells, with the remaining cells derived from NG2⁺ pericytes.⁹⁰ Therefore, adventitial perivascular Gli1⁺ cells may be the most primitive MSCs that act as mesenchymal precursors in mouse incisor. NG2⁺ pericytes represent a MSC subpopulation derived from true MSCs (Gli1⁺ cells). Following injury, pericytes might be activated, leading to the promotion of tissue repair through differentiation into mesenchymal cells.⁵⁰

Myofibroblast sources: tissue-resident MSCs

Differential gene expression patterns were observed between MSCs in several organs. BM MSCs showed markedly higher expression in signal transducer and activator of transcription 2 (STAT2), transforming growth factor-β receptor (TGFβR) 2, fibroblast growth factor (FGF)-18, and retinoic acid receptor-α (RARAα) compared with MSCs from other tissues. The expression of insulin-like growth factor-2 (IGF-2), jagged-1 (JAG1), bone morphogenic protein-2 (BMP2), FGF-13, and angiopoietin-like-1 (ANGPTL1) was predominantly found in MSCs of skeletal muscle.

The expression of TGFβR3, PDGFRα, WNT-inducible signaling protein-1 (WISP1), IL-7, osteoglycin (OGN), IGF-1, and suppressor of cytokine signaling-5 (SOCS5) was predominantly found in the periosteum.¹³³ This indicates that MSCs have different gene expression profiles depending on the resident tissue in vivo. Genetic labeling techniques revealed tissue-resident MSC populations in vivo (Table 1). This technique showed that resident MSC pools may include perivascular MPCs and that MPCs can possess pericyte features.¹³⁴

Table 1.

Mesenchymal stromal cell populations contributing to tissue fibrosis

Organ	Isolation Marker	Animal	MSC Population	Contribution to Mesenchymal Tissue	References
Bone marrow	LepR	Mice	Perivascular MSCs	Myofibroblast, osteocyte, chondrocyte, adipocyte	⁵³
Bone marrow	Gli 1	Mice	Perivascular MSC-like cells	Myofibroblasts	⁵⁰
Lung	FoxD1	Mice	Pericyte, resident fibroblasts	Myofibroblasts	¹¹²
	NG2	Mice	Pericyte-like cells?	Myofibroblasts?, resident fibroblasts	¹¹³
	TBX4	Mice	MSC-like cells	Fibroblasts, smooth muscle cells, pericytes, endothelial cells	¹¹⁴
	ABCG2	Mice	Perivascular mesenchymal stem cells	Myofibroblasts	¹¹⁵
	Gli1	Mice	Perivascular MSC-like cells	Myofibroblasts	⁵⁰
	LPA1	Mice	Resident MSC-like cells	Myofibroblasts	¹¹⁶
Kidney	FoxD1	Mice	Perivascular MSC-like cells, pericyte, peritubular fibroblasts	Pericytes, myofibroblasts	¹¹⁷
Kidney	Gli1	Mice	Perivascular MSC-like cells	Myofibroblasts	⁵⁰
Heart	TCF21	Mice	Resident fibroblasts	Myofibroblasts	¹¹⁸
Heart	Gli1	Mice	Perivascular MSC-like cells	Myofibroblasts	⁵⁰
Skin	ADAM12	Mice	Perivascular MPC	Myofibroblasts	¹⁰⁸
	EN-1	Mice	CD26/DPP4-positive fibroblast	Myofibroblasts	¹¹⁹
	Dlk1	Mice	Reticular fibroblasts, adipocytes, preadipocytes	Myofibroblasts	¹⁰⁷

ABCG2, ATP-binding cassette sub-family G member 2; ADAM12, A disintegrin and metalloproteinase domain 12; Dlk1, delta-like homolog 1; DPP4, dipeptidyl peptidase-4; EN-1, engrailed-1; FoxD1, forkhead box D1; Gli1, glioma-associated oncogene homolog 1; LepR, leptin receptor; LPA1, lysophosphatidic acid 1; MPC, mesenchymal progenitor cell; MSC, mesenchymal stromal cell; NG2, neural/glial antigen 2; TBX4, Tbox gene 4; TCF21, transcription factor 21.

These findings indicate that resident MSC pools may include perivascular MPCs that give rise to specific connective tissue cell types.¹³⁵ Further study suggests that pericytes represent a source of MSCs throughout the body.¹³⁶ Sacchetti et al. demonstrated that pericytes isolated from human organs gave rise to tissue-specific MSC cultures in vitro.¹³³ These MSC cultures could differentiate into osteoblasts only if they were derived from BM. Therefore, pericytes distributed in different tissues might provide a local source of tissue-specific MSCs.

One possible mechanism of tissue-specific MSC formation is that precursor pericytes give rise to tissue-specific pericytes, which finally generate the tissue-specific MSCs.⁹⁸ Yianni and Sharpe reported on pericyte-derived MSCs (pMSCs) and proposed that pMSCs can migrate to an area of injury and differentiate into tissue-specific cells for repair¹³⁷ (Fig. 1).

Figure 1.

A model of perivascular MSC response to injury in tissue repair. In homeostasis (left), MSCs might behave as progenitor cells giving rise to vascular components (e.g., smooth muscle cells and pericytes) and surrounding stromal cells, such as adipocytes. Following injury (right), pericytes can detach from the vessel wall and act as MSCs, which Yianii and Sharpe referred to as pMSCs. pMSCs can proliferate and move to the area of injury where pMSCs differentiate into tissue-specific cells to promote repair, along with MSCs that are not pericyte derived.^137,155 MSC, mesenchymal stromal cell; pMSC, pericyte-derived MSC. Color images are available online.

In this process, differentiation into pMSCs might depend on their tissue residence, possibly attributable to niche-specific signals or intrinsic mechanisms for selection of the appropriate cell population.¹³³ However, Sinha et al. recently demonstrated that BM-derived myeloid cells play an important role, contributing up to two-thirds of fibroblasts in granulation tissues. This suggests that pericytes are a quantitatively small component of progenitor cells that give rise to granulation tissue fibroblasts in wound healing.¹³⁸

The activation of myofibroblast progenitors is an essential step in fibrosis and, thus, identifying the cellular source of myofibroblasts is important. Perivascular cells and MSC-like cells have recently been proposed as a source of myofibroblasts in fibrotic disease.⁹⁹ Based on the findings that MSCs contribute to the formation of granulation tissues through their immunomodulatory functions, MSCs have been shown to attenuate fibrosis by decreasing the number of myofibroblasts in the later phase of healing.⁵⁸ However, Ding et al. demonstrated that BM-MSCs may activate the profibrotic profile of deep dermal fibroblasts.¹³⁹

Pericytes have been suggested to be the primary source of scar-forming fibroblasts.¹⁴⁰ The role of pericytes as myofibroblast precursors has been indicated by studies of fibrogenesis in several organs. Previous studies of fibrogenesis of liver, kidney, and systemic sclerosis suggested that pericytes are myofibroblast precursors.^141
–143

The concept of pericyte to myofibroblast transition has been proposed by several groups in relation to fibrogenesis in organs.^103,144 Pericytes in perivascular regions are activated by a combination of cytokines, such as PDGFRβ, vascular endothelial growth factor (VEGF)-A, and TGFβ1, during the process of fibrosis. Pericytes may then exhibit changes in marker profiles, detach from the vessel basement membrane, and transform into myofibroblasts, resulting in fibrosis and scar formation¹⁴⁵ (Fig. 1). They further demonstrated that PDGFRβ⁺ pericytes contribute to scar formation and express fibroblast-associated markers.¹⁴⁶

Analysis of forkhead box D1 (FoxD1)-Cre mice revealed that PDGFRβ⁺/CD73⁺ mesenchymal cells are a candidate myofibroblast precursor possibly derived from pericytes.¹⁴⁷ A unilateral ureteral obstruction kidney fibrosis model in collagen type I, α1 reporter mice demonstrated that snail homolog 1 (Snail1) and Id1 transcription induces the differentiation of pericytes into myofibroblasts.¹⁰³

Further analysis of double-transgenic Nestin-GFP/NG2-DsRed mice demonstrated that type 1 pericytes accumulated in blood vessels and produced collagen in skeletal muscle and in bleomycin-induced pulmonary fibrosis, but not in renal fibrosis and cardiac fibrosis, indicating that pericytes react differently to injury depending on the organ affected.¹⁴⁸ Therefore, pericytes may generate tissue-specific myofibroblasts to promote tissue fibrosis.

The levels of αSMA expression for identification of myofibroblasts vary at different stages of contraction and depend on the condition of the cell in which it is located. Therefore, a quantitative estimation of αSMA expression levels is needed to investigate the role of pericytes as a source of myofibroblasts in wound healing. It must be noted that tissue fibrosis in the early phase of healing contributes to the promotion of tissue stiffness, leading to further force generation. However, excessive fibrosis in the later phase of healing is disruptive to a patient's mobility and cosmetics.

Skin MSCs reside in distinct layers of the dermis and contribute to the formation of skin connective tissues. Previous analysis in mice demonstrated that the delta-like homolog 1 (Dlk1)⁺/Sca-1⁺ and Dlk1⁻/Sca-1⁺ MSC populations are the cell origin of reticular fibroblasts, preadipocytes, and adipocytes in the hypodermis.¹⁴⁹ Dlk1⁺ MSCs become activated and generate myofibroblasts, leading to dermal fibrosis in wound repair. In contrast, perivascular dermal MSCs in mice were found to express A disintegrin and metalloproteinase domain 12 (ADAM12) (Fig. 2).

Figure 2.

Mechanisms of dermal fibrosis induced by tissue-specific resident MSCs in mice. Papillary dermal CD26⁺/Sca-1⁻ fibroblasts might contribute to formation in upper dermis connective tissues, whereas Dlk1⁺/Sca-1⁺ and Dlk1⁻/Sca-1⁺ MSCs residing in the hypodermis can lead to generation of reticular fibroblasts, preadipocytes, and adipocytes. Dlk1⁺ MSCs might generate αSMA⁺ myofibroblasts, leading to production and deposition of extracellular matrix in the dermis during the healing process. ADAM12⁺ MSCs residing in perivascular spaces can become activated following injury and generate αSMA⁺ myofibroblasts.^149,150 ADAM12, a disintegrin and metalloproteinase domain 12; Dlk1, delta-like homolog 1; ECM, extracellular matrix; Sca-1, stem cell antigen-1; SMA, smooth muscle actin. Color images are available online.

Similar to Dlk1⁺ MSCs, ADAM12 ⁺ perivascular MSCs are fibrogenic progenitors and proliferate to generate αSMA⁺ myofibroblasts in response to injury, leading to dermal fibrosis during healing.¹⁵⁰ Therefore, Dlk1⁺ MSCs and ADAM12⁺ MSCs might be major contributors of extracellular matrix production in dermal fibrosis of mice following injury. ADMA12 was previously shown to be expressed in myofibroblast progenitors in the perivascular region of mouse skin.¹⁵⁰

In human, ADAM12 was found to be highly upregulated in BM-MSCs.¹⁵¹ Furthermore, a treatment with TGFβ1 upregulated the expression of ADAM12 in the perivascular cells of diffuse cutaneous systemic sclerosis patients.¹⁵² Collectively, these findings suggest that resident MSC-like cells in human skin predominately express ADAM12. The ablation of ADAM12⁺ cells was also shown to attenuate fibrosis in mouse skin.¹⁵⁰ Therefore, the ablation of ADAM12⁺ cells in human skin may lead to the development of novel antifibrosis therapies for patients.⁹⁹

Future Directions

Perivascular investigations may lead to the development of treatments for tissue fibrosis. Zhang et al. demonstrated that oral curcumin delivery in carbon tetrachloride-exposed rats inhibited organ fibrosis and vascularization through the transrepression of PDGFRβ.¹⁵³ Lin et al. demonstrated that circulating soluble receptor ectodomains attenuated kidney fibrosis and capillary rarefaction through blockade of either PDGFRβ signaling in pericytes or vascular endothelial growth factor receptor 2 (VEGFR2) signaling in endothelial cells.¹⁵⁴ Therefore, targeting growth factor receptors expressed in pericytes may contribute to inhibition of fibrosis in organs.¹³⁰

The potential of pericytes to differentiate into several cell types has been established by many studies, and the ability of pericytes to differentiate into tissue-specific cells is acknowledged.

To clarify the precise role of pericytes as a source of myofibroblasts in tissue fibrosis, genetically engineered animals, such as knockout or transgenic animals need to be developed; however, the lack of specific markers of pericytes may hinder investigations on the phenotypic plasticity of pericytes during the fibrotic process. Adipose tissue MSCs have been found to be available in the treatment of soft tissue defect and skin wound healing. Autologous adipose tissue MSCs combined with cytokines and biomaterials will be an alternative method for the treatment of damaged tissues.

Summary

Recent advanced technique led to the characterization of perivascular MSC populations and pericytes might provide local source of tissue-specific MSCs, which can differentiate into tissue-specific cells for repairing and fibrosis. The clarification of the roles of perivascular MSCs may bring new approaches for the treatment of fibrogenic disease and perivascular MSCs have the potential to be used as a target of antifibrosis and a cell therapy for wound healing. Autologous adipose tissue MSCs combined with cytokines and biomaterials is effective in the treatment of soft tissue defect and skin wound healing.

Take-Home Messages

MSCs can be isolated from various tissues and functional differences are found between populations of MSCs from different sources.

Perivascular MSCs may contain progenitor cells capable of differentiating to specific tissue cells.

Analysis of MSC activity can lead to the development of more effective therapies for the treatment of fibrogenic disease.

Autologous adipose tissue MSCs combined with cytokines and biomaterials play an important role in regeneration of damaged tissues.

Footnotes

Author Confirmation

The article has not been published previously, that it is not under consideration for publication elsewhere, and that, if accepted, it will not be published elsewhere in the same form without the written consent of the publisher.

Acknowledgments and Funding Sources

The author thanks Kayo Tsuburaya (Department of Pathology, School of Medicine, Toho University, Tokyo, Japan) for assistance in literature research. He thanks Ryoichi Mori (Department of Investigative Pathology, Graduate School of Biomedical Sciences, Nagasaki University) for informational support. Wound healing research in their laboratory was supported by the Japanese Society for the Promotion of Science KAKENHI grants JP17K11555 and JP19K10017 and the Strategic Research Foundation Grant-aided Project for Private School grants S1101016 and S1411015 from the Ministry of Education, Culture, Sports, Science, and Technology of Japan.

Author Disclosure and Ghostwriting

There are no conflicts of interest. No ghostwriter was used for this article.

About the Author

Yoshikiyo Akasaka, MD, PhD is a pathologist in Department of Pathology, Toho University School of Medicine. He is also a full professor in Research Center, Toho University Graduate School of Medicine, Japan, and president of the Japanese Society for Wound Healing (JSWH).

Abbreviations and Acronyms

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