Human endometrial perivascular stem cells exhibit a limited potential to regenerate endometrium after xenotransplantation

Abstract

STUDY QUESTION What are the localization, characteristics and potential for tissue regeneration of two perivascular stem cells, namely CD34+ adventitial cells and CD146+ pericytes, in human endometrium? SUMMARY ANSWER Human endometrial CD34+ adventitial cells (located in the outermost layer of blood vessels and mainly in the basal layer) and CD146+ pericytes showed mesenchymal stem cell (MSC) phenotypes in in vitro culture, but presented limited potential to regenerate endometrium. WHAT IS KNOWN ALREADY Periodic endometrial regeneration is considered to be maintained by MSCs. Blood vessel wall, regarded as stem cell niche, harbors a large reserve of progenitor cells that may be integral to the origin of MSCs. However, a lack of validated markers has hampered the isolation of putative endometrial MSCs. Currently, CD146+ pericytes and Sushi Domain Containing 2 (SUSD2) positive cells have been identified in the endometrial perivascular region as sharing MSCs characteristics. STUDY DESIGN, SIZE, DURATION The locations of adventitial cells and pericytes in the human endometrium were identified by immunofluorescence staining (n = 4). After CD34+CD146−CD45−CD56−CD144− adventitial cells and CD146+CD34−CD45−CD56−CD144− pericytes were isolated from the endometrium of normal women (n = 6) by fluorescence-activated cell sorting, their characteristics were investigated in culture. Adventitial cells and pericytes were induced to differentiate, respectively, into vascular endothelial-like cells or endometrial stromal-like cells in vitro, with their potential explored by in vivo xenotransplantation (n = 2 in each group) and eutopic transplantation (n = 2 in each group). PARTICIPANTS/MATERIALS, SETTING, METHODS CD34+ adventitial cells and CD146+ pericytes were cultured in the inducing medium to differentiate into endothelial-like cells in vitro, and then analyzed for CD31, von Willebrand factor immunofluorescent staining and tube formation. They were also cultured to differentiate into endometrial stromal cells in vitro, with the expression of vimentin and CD13 being detected by western blot before and after induction, and the expression of prolactin and insulin-like growth factor-binding protein 1 being determined as well. Single dispersed CD34+ adventitial cells and CD146+ pericytes were respectively transplanted under the kidney capsule of NOG mice to investigate their differentiation potential in vivo. A eutopic transplantation model was constructed by grafting recellularized uterine matrix loaded up with CM-Dil labeled adventitial cells or pericytes into the injury region of nude rat’s uterus. MAIN RESULTS AND THE ROLE OF CHANCE CD34+ adventitial cells were mainly located at the outmost layer of endometrial large vessels, while CD146+ pericytes were found surrounding the inner endothelial cells of microvessels. A small proportion of CD34+ adventitial cells expressed SUSD2. The number of adventitial cells was ∼40 times higher than that of pericytes in the endometrium. Both adventitial cells and pericytes showed MSC phenotypes after in vitro culture. After in vitro induction into endometrial endothelial-like cells and stromal-like cells, adventitial cells showed higher plasticity than pericytes and a closer correlation with stromal-like cells. In the mouse xenotransplantation model, vimentin+ cells, CD31+ endothelial-like cells and CD146+ pericyte-like cells could be observed after adventitial cells were transplanted. CM-Dil-labeled adventitial cells or pericytes could survive in the immunocompromised nude rats after eutopic transplantation, and vimentin+ cells were detected. In addition, CM-Dil-labeled adventitial cells or pericytes did not express α-smooth muscle actin or E-cadherin after transplantation LARGE SCALE DATA N/A LIMITATIONS, REASONS FOR CAUTION CD34 was chosen as a novel marker to isolate adventitial cells from human endometrium according to previous literature. The association of endometrial CD34+ adventitial cells and SUSD2+ MSCs should be further investigated. WIDER IMPLICATIONS OF THE FINDINGS The decellularized uterine matrix model might be useful in endometrial stem cell therapy. STUDY FUNDING/COMPETING INTEREST(S) L.D. is supported by grants from National Key Research and Development Program of China (2018YFC1004700), Nature Science Foundation of China (81871128, 81571391) and Nanjing Medical Science Development Project (ZKX16042). H.S. is supported by a grant from Jiangsu Province Social Development Project (BE2018602). X.Z. was supported by grants from the Postgraduate Innovative Project of Jiangsu Province (KYCX19-1177). The authors declare no conflict of interest.

Stem cell-based therapy for ameliorating intrauterine adhesion and endometrium injury

“…Restructuring and maturation of the vascular network can facilitate embryo implantation [ 81 ]. Blood vessel walls are considered as stem cell-niche with a large reservoir of progenitor cells [ 82 ]. Previous study has found that eMSCs in the basalis and functionalis are mainly perivascular cells, including CD146 + and CD140b + (platelet-derived growth factor-β, PDGFR-β +) pericytes [ 50 , 51 ], sushi domain-containing-2 + (SUSD2 + ) cells [ 83 ] and CD34 + adventitial cells (located in the outermost layer of blood vessels and mainly in the basal layer) [ 82 ].…”

Endometrial Perivascular Progenitor Cells and Uterus Regeneration

“…Crisan et al have shown that both CD146 and PDGF-Rβ are ubiquitously expressed on pericytes surrounding endothelial cells in the capillaries of all human organs, and the co-expression of NG2 and deficient expression of endothelial markers (CD144, CD31, CD34, vWF, and UEA-1) were further confirmed by immunodetection [46]. CD146 + pericytes mainly reside in capillaries and microvessels in both the functionalis and basalis of the endometrium (Figure 2a) [47]. Moreover, higher clonogenicity was observed in CD146 + PDGF-Rβ + cells expressing SUSD2 (or W5C5), which in vivo constitute endometrial stromal tissues [48].…”

“…Interestingly, CD34 + adventitial cells express pericyte markers such as CD146, PDGFR-β, α-SMA, and NG2 after exposure to angiopoietin-2, suggesting that adventitial cells are the precursors of pericytes [57]. Recently, CD34 + CD146 − CD45 − CD56 − CD144 − adventitial cells have also been proven for the first time to exist in human endometrial tissues and are located in the outermost layer of large vessels, particularly the basal layer (Figure 2b), with an abundance nearly 40 times higher than that of pericytes [47]. Furthermore, SUSD2, another marker of perivascular cells that possesses MSC properties, is expressed mostly in the tunica media of vessels and in only a small proportion of CD34 + adventitial cells (Figure 2c).…”

“…Furthermore, SUSD2, another marker of perivascular cells that possesses MSC properties, is expressed mostly in the tunica media of vessels and in only a small proportion of CD34 + adventitial cells (Figure 2c). After inducing culture, both CD34 + adventitial cells and CD146 + pericytes derived from the human endometrium, could differentiate into endometrial stromal-like cells, while the expression levels of vimentin and CD13 significantly increased in adventitial cells [47].…”

“…It has been documented that CD146 + pericytes from the human endometrium could be utilized in uterine regeneration in rat models that underwent 1.5 cm × 0.5 cm full-thickness uterine resection, with a focus on expounding the capacity of in vitro angiogenesis and in vivo neovascularization by pericytes, which is probably induced by secretion of CYR61 [86]. Moreover, when induced on Matrigel for tube formation analysis, CD34 + adventitial cells formed branches at an earlier stage than CD146 + pericytes, and later developed a network loop structure that positively stained for CD31 and vWF [47]. Nevertheless, whether CD34 + adventitial cells embody a more compelling angiogenetic capacity in vivo in injured tissues, particularly in an AS uterus, and whether perivascular cells contribute to tissue regeneration through the angiogenetic process remains unclear.…”