Humans exhibit sex differences in various physiologic and pathologic conditions. Several diseases associated with SMC dysfunction show sex difference, including atherosclerosis , abdominal artery aneurysm , and stroke . Previously, these sex differences were mainly attributed to the differential regulatory effects of the gonadal hormones. However, there is accumulating evidence that sex differences might not relate only to variations of circulating sex steroid levels, but also to the intrinsic differences of the target cell . Functional SMCs have great therapeutic potential for restoring organ function and promoting tissue regeneration in the aforementioned diseases through transplantation or integration into tissue-engineered vessels [39, 40]. Little is known about whether sex differences exist in the differentiation of stem cells to the mesenchymal cell lineage and in the function of human pSMCs and SMCs resulting from this differentiation. In this study, using a standardized and clinical-grade differentiation protocol, we demonstrated that male and female hPSCs can be efficiently differentiated into pSMCs in vitro with nearly equal differentiation efficiencies. However, sex differences exist in the function and proliferation of hPSC-derived pSMCs in response to E2 stimulation.
It has been reported that stem cells derived from different tissue sources show marked variability in their ability to differentiate into pancreatic cells and cardiomyocytes . To examine whether cell sex affects stem cell differentiation, we compared differentiation efficiency between male and female hPSCs using the same smooth muscle differentiation protocol. CD34+/CD31+ cells are known to give rise to vessels during embryonic development  and have been identified as VPCs which can generate SMCs via in-vitro directed differentiation [12, 43]. Therefore, the percentage of CD34+/CD31+ VPCs in the hPSC derivatives was used to compare SMC differentiation efficiency in male and female hPSCs. Our data show that both male and female hPSCs can be induced into VPCs with similar efficiencies, suggesting that cell sex has no effect on the pSMC-committed differentiation. This observation is in agreement with recent studies [44, 45] which, although not directly focusing on the role of cell sex in the stem cell differentiation, showed that SMCs can be efficiently generated from male and female hPSCs through similar differentiation protocols as ours.
Stem cell differentiation in sexually dimorphic mammalian organs (such as sexual glands) is regulated by sex hormone [38, 46]. An important question for translation of stem cell-based therapies is whether the differentiation of stem cells in organs that do not show sex-specific morphological differences, such as various hollow organs, is also influenced by circulating sex hormones. The role of E2 in the function, proliferation, and transdifferentiation of mature SMCs has been studied extensively. For example, E2 mediates airway SMC contraction by modulating intracellular Ca2+  and an anti-proliferative effect of nitric oxide in female vascular SMCs . Exogenous E2 induces the expression of α-actin in aortic SMCs and abolishes progressive growth of aortic aneurysms in female ovariectomized mice . However, the effect of sex hormone on the differentiation of pSMCs has not been reported. The current study shows that exposure to E2 during the intermediate steps in the differentiation from hPSC to SMCs promotes gene expression of myogenic markers only in female hPSC-derived pSMCs. This is consistent with female animal data demonstrating that E2 treatment can promote the differentiation of female rat wall-resident CD34+ progenitor cells into vascular SMCs . Our data also revealed that E2 treatment during the terminal differentiation step from pSMCs to SMCs did not affect gene expression of myogenic makers, suggesting that optimization of stem cell therapies for SMC with hormonal manipulation should be targeted at the intermediate stage of SMC differentiation. The lack of effect on SMC markers in the terminally differentiated SMCs may be due to saturation of these markers in the mature SMCs.
SMCs modulate ECM metabolism in tissues through synthesis of several types of ECM components (such as collagen I, collagen III, collagen IV, and fibronectin) and matrix remodeling enzymes such as matrix metalloproteinases (MMPs) and their inhibitors, the tissue inhibitors of MMPs (TIMPs) [31, 32]. MMP-1 and MMP-2 are capable of degrading multiple ECM components, including collagen and elastin . Generally, all TIMPs are capable of inhibiting all known MMPs and ECM proteolysis. However, the efficacy of MMP inhibition varies with each TIMP. TIMP-1 is a strong inhibitor of many MMPs, except for some of the membrane type MMPs, while TIMP-2, TIMP-3, and TIMP-4 interact with pro-MMP-2. Elevated MMP-1 and MMP-2 have been strongly associated with ruptured atherosclerotic plaques and aortic aneurysms . Thus, SMCs are important in maintaining the ECM homeostasis in hollow organs . In this study, we identified that female pSMCs had a significantly lower expression of MMP-1 gene and a significantly higher expression of collagen I gene compared to male pSMCs. Furthermore, E2 stimulation decreases MMP-2 activity and increases TIMP-1 expression. Taken together, these findings suggest that female pSMCs may inhibit ECM proteolysis in an estrogen environment compared to male pSMCs. These differential cell responses to estrogen could contribute to the observed differences in cardiovascular morbidity between men and women, and can be optimized to enhance efficacy of cell replacement therapy and tissue-engineering applications. We note that MMP activity in the cell culture supernatants was analyzed by gelatin zymography. Although gelatin zymography is an excellent and simple tool for the identification of MMP activity, it is a semiquantitative technique  and does not reveal the integrated gelatinase activity in the cell culture media. Therefore, future in-vivo cell transplantation studies are needed to more accurately delineate the sex differences of pSMCs in ECM modulation.
Ability to generate a sufficient number of functional pSMCs is important for the translation of pSMC-based therapy. Using time-lapse microscopy, we found that female pSMCs proliferate more frequently than the corresponding male pSMCs. This observation is consistent with previous studies involving other stem/progenitor cell types [25, 54]. Another observation from our cell proliferation analysis is that the number of mitosis events in female HuF5-iPSC-pSMCs was significantly higher than that in female H9-ESC-pSMCs. Variability between different lines is likely due to intrinsic differences between individuals or to persistent epigenetic modifications in the iPSC lines. We do not believe the increase in proliferation rate in female iPSC-derived pSMCs is associated with persistent expression of pluripotency genes (POF5F1, KLF4, SOX2, and c-MYC) from the retroviral vector reprogramming technique, because our previous study showed that the FACS purification step for VPCs yields >99.9% purity with undetectable levels of these genes by PCR (in press). The undetectable but potential subpopulation of cells with residual pluripotency genes in the differentiated population would be too low to account for the significant increases in proliferation rates.
Our current study also showed that E2 affects proliferation of pSMCs in a sex-dependent manner. While female pSMCs exhibited higher overall mitotic events compared to male pSMCs, E2 stimulation only increased mitotic events in male pSMCs. It is interesting to note that while estrogen increased mitosis of male pSMCs, E2 treatment did not enhance the differentiation of male pSMCs from the VPC intermediate. Published data suggest that E2 can have different effects on CD34+ progenitor cells [50, 55]; it can accelerate the proliferation of undifferentiated CD34+ progenitor cells, while promoting further differentiation in differentiating progenitor cells. Thus, it is possible that there was a relatively higher fraction of undifferentiated VPCs that persisted in the male pSMC population compared to female pSMCs after FACS sorting. E2 stimulation of this small subpopulation increased proliferation, but did not increase differentiation from VPCs to pSMCs. Future studies involving detailed characterization of small subpopulations in the FACS sorted VPCs will help elucidate this possible effect.
Another possible confounder is that hPSCs can undergo genetic changes during in-vitro culture  which could affect proliferation. These changes have been documented in the H1/9 ESC lines . Because these genetic aberrations are difficult to detect with karyotyping, a higher resolution technique is required to examine this source of error. Given the limited and exploratory nature of our studies, we opted to correlate the hESC data with data from iPSC lines, and found a similar pattern in both groups. Future studies are necessary to confirm the nature differential proliferation between sexes.
ER-α and ER-β are expressed in the early development embryoid bodies and modulate the differentiation of hESCs into various cell types . There are robust data showing that physiologic levels of E2 can stimulate proliferation of hESCs, confirming an end-effect through interaction with ERs. We found that ER-α and ER-β were robustly expressed in the iPSC-derived pSMCs. Based on our limited sample size, it is not possible to determine whether there is differential expression of ER-α compared to ER-β. Data on the relative distribution of these receptors in human PSCs is scant due to limited available lines. Most studies document expression of these receptors, rather than relative expression. ER-β is also known to be involved in multiple aspects of SMC function and development, such as contraction, maturation, proliferation, and migration [58, 59]. It is possible that the effects observed in this study are the result of differential expression of ERs. Additional loss-of-function studies are needed to investigate the role of differential ER expression in modulating proliferation of pSMCs.
Finally, in-vivo animal studies are needed to confirm whether the observed cell-sex differences in pSMCs would affect the efficiency of cell therapy for conditions involving SMCs.