A comparative assessment of adipose‐derived stem cells from subcutaneous and visceral fat as a potential cell source for knee osteoarthritis treatment

The intra‐articular injection of adipose‐derived stem cells (ASCs) is a novel potential therapy for patients with osteoarthritis (OA). However, the efficacy of ASCs from different regions of the body remains unknown. This study investigated whether ASCs from subcutaneous or visceral adipose tissue provide the same improvement of OA. Mouse and human subcutaneous and visceral adipose tissue were excised for ASC isolation. Morphology, proliferation, surface markers and adipocyte differentiation of subcutaneous ASCs (S‐ASCs) and visceral ASCs (V‐ASCs) were analysed. A surgically induced rat model of OA was established, and 4 weeks after the operation, S‐ASCs, V‐ASCs or phosphate‐buffered saline (PBS, control) were injected into the articular cavity. Histology, immunohistochemistry and gene expression analyses were performed 6 weeks after ASC injection. The ability of ASCs to differentiate into chondrocytes was assessed by in vitro chondrogenesis, and the immunosuppressive activity of ASCs was evaluated by co‐culturing with macrophages. The proliferation of V‐ASCs was significantly greater than that of S‐ASCs, but S‐ASCs had the greater adipogenic capacity than V‐ASCs. In addition, the infracted cartilage treated with S‐ASCs showed significantly greater improvement than cartilage treated with PBS or V‐ASCs. Moreover, S‐ASCs showed better chondrogenic potential and immunosuppression in vitro. Subcutaneous adipose tissue is an effective cell source for cell therapy of OA as it promotes stem cell differentiation into chondrocytes and inhibits immunological reactions.     

Characterization and comparison of adipose tissue‐derived cells from human subcutaneous and omental adipose tissues

Different fat depots contribute differently to disease and function. These differences may be due to the regional variation in cell types and inherent properties of fat cell progenitors. To address the differences of cell types in the adipose tissue from different depots, the phenotypes of freshly isolated adipose tissue‐derived cells (ATDCs) from subcutaneous (SC) and omental (OM) adipose tissues were compared using flow cytometry. Our results showed that CD31^?CD34⁺CD45^?CD90^‐CD105^?CD146⁺ population, containing vascular smooth muscle cells and pericytes, was specifically defined in the SC adipose tissue while no such population was observed in OM adipose tissue. On the other hand, CD31^?CD34⁺CD45^?CD90^?CD105^?CD146^? population, which is an undefined cell population, were found solely in OM adipose tissue. Overall, the SC adipose tissue contained more ATDCs than OM adipose tissue, while OM adipose tissue contained more blood‐derived cells. Regarding to the inherent properties of fat cell progenitors from the two depots, adipose‐derived stem cells (ADSCs) from SC had higher capacity to differentiate into both adipogenic and osteogenic lineages than those from OM, regardless of that the proliferation rates of ADSCs from both depots were similar. The higher differentiation capacity of ADSCs from SC adipose tissue suggests that SC tissue is more suitable cell source for regenerative medicine than OM adipose tissue. Copyright © 2009 John Wiley & Sons, Ltd.     

Ex vivo organ culture of adipose tissue for in situ mobilization of adipose‐derived stem cells and defining the stem cell niche

In spite of the advances in the knowledge of adipose‐derived stem cells (ASCs), in situ location of ASCs and the niche component of adipose tissue (AT) remain controversial due to the lack of an appropriate culture system. Here we describe a fibrin matrix‐supported three‐dimensional (3D) organ culture system for AT which sustains the ASC niche and allows for in situ mobilization and expansion of ASCs in vitro. AT fragments were completely encapsulated within the fibrin matrix and cultured under dynamic condition. The use of organ culture of AT resulted in a robust outgrowth and proliferation in the fibrin matrix. The outgrown cells were successfully recovered from fibrin by urokinase treatment. These outgrown cells fulfilled the criteria of mesenchymal stem cells, adherence to plastic, multilineage differentiation, and cell surface molecule expression. In vitro label retaining assay revealed that newly divided cells during the culture resided in interstitium between adipocytes and capillary endothelial cells. These interstitial stromal cells proliferated and outgrew into the fibrin matrix. Both in situ mobilized and outgrown cells expressed CD146 and α‐smooth muscle actin (SMA), but no endothelial cell markers (CD31 and CD34). The structural integrity and spatial approximation of CD31^?/CD34^?/CD146⁺/SMA⁺ interstitial stromal cells, adipocytes, and capillary endothelial cells were well preserved during in vitro culture. Our results suggest that ASCs are natively associated with the capillary wall and more specifically, belong to a subset of pericytes. Furthermore, organ culture of AT within a fibrin matrix‐supported 3D environment can recapitulate the ASC niche in vitro. J. Cell. Physiol. 224: 807–816, 2010. © 2010 Wiley‐Liss, Inc.     

Larger anti-adipogenic effect of angiotensin II on omental preadipose cells of obese humans

Objective: The ability to form new adipose cells is important to adipose tissue physiology; however, the mechanisms controlling the recruitment of adipocyte progenitors are poorly understood. A role for locally generated angiotensin II in this process is currently proposed. Given that visceral adipose tissue reportedly expresses higher levels of angiotensinogen compared with other depots and the strong association of augmented visceral fat mass with the adverse consequences of obesity, we studied the role of angiotensin II in regulating adipogenic differentiation in omental fat of obese and non‐obese humans. Research Methods and Procedures: The angiotensin II effect on adipose cell formation was evaluated in human omental adipocyte progenitor cells that were stimulated to adipogenic differentiation in vitro. The adipogenic response was measured by the activity of the differentiation marker glycerol‐3‐phosphate dehydrogenase. Results: Angiotensin II reduced the adipogenic response of adipocyte progenitor cells, and the extent of the decrease correlated directly with the subjects’ BMI (p = 0.01, R² = 0.30). A 56.3 ± 3.4% and 44.5 ± 2.7% reduction of adipogenesis was found in obese and non‐obese donors’ cells, respectively (p < 0.01). The effect of angiotensin II was reversed by type 1 angiotensin receptor antagonist losartan. Discussion: A greater anti‐adipogenic response to angiotensin II in omental adipose progenitor cells from obese subjects opens a venue to understand the deregulation of visceral fat tissue cellularity that has been associated with severe functional abnormalities of the obese condition.     

Fat depot-specific gene signature and ECM remodeling of Sca1high adipose-derived stem cells

Stem cell antigen-1 (Sca1 or Ly6A/E) is a cell surface marker that is widely expressed in mesenchymal stem cells, including adipose-derived stem cells (ASCs). We hypothesized that the fat depot-specific gene signature of Sca1^high ASCs may play the major role in defining adipose tissue function and extracellular matrix (ECM) remodeling in a depot-specific manner. Herein we aimed to characterize the unique gene signature and ECM remodeling of Sca1^high ASCs isolated from subcutaneous (inguinal) and visceral (epididymal) adipose tissues. Sca1^high ASCs are found in the adventitia and perivascular areas of adipose tissues. Sca1^high ASCs purified with magnetic-activated cell sorting (MACS) demonstrate dendrite or round shape with the higher expression of cytokines and chemokines (e.g., Il6, Cxcl1) and the lower expression of a glucose transporter (Glut1). Subcutaneous and visceral fat-derived Sca1^high ASCs particularly differ in the gene expressions of adhesion and ECM molecules. While the expression of the major membrane-type collagenase (MMP14) is comparable between the groups, the expressions of secreted collagenases (MMP8 and MMP13) are higher in visceral Sca1^high ASCs than in subcutaneous ASCs. Consistently, slow but focal MMP-dependent collagenolysis was observed with subcutaneous adipose tissue-derived vascular stromal cells, whereas rapid and bulk collagenolysis was observed with visceral adipose tissue-derived cells in MMP-dependent and -independent manners. These results suggest that the fat depot-specific gene signatures of ASCs may contribute to the distinct patterns of ECM remodeling and adipose function in different fat depots.     

Human adipose CD34+CD90+ stem cells and collagen scaffold constructs grafted in vivo fabricate loose connective and adipose tissues

Stem cell based therapies for the repair and regeneration of various tissues are of great interest for a high number of diseases. Adult stem cells, instead, are more available, abundant and harvested with minimally invasive procedures. In particular, mesenchymal stem cells (MSCs) are multi‐potent progenitors, able to differentiate into bone, cartilage, and adipose tissues. Human adult adipose tissue seems to be the most abundant source of MSCs and, due to its easy accessibility; it is able to give a considerable amount of stem cells. In this study, we selected MSCs co‐expressing CD34 and CD90 from adipose tissue. This stem cell population displayed higher proliferative capacity than CD34^?CD90^? cells and was able to differentiate in vitro into adipocytes (PPARγ⁺ and adiponectin⁺) and endothelial cells (CD31⁺VEGF⁺Flk1⁺). In addition, in methylcellulose without VEGF, it formed a vascular network. The aim of this study was to investigate differentiation potential of human adipose CD34⁺/CD90⁺ stem cells loaded onto commercial collagen sponges already used in clinical practice (Gingistat) both in vitro and in vivo. The results of this study clearly demonstrate that human adult adipose and loose connective tissues can be obtained in vivo, highlighting that CD34⁺/CD90 ASCs are extremely useful for regenerative medicine. J. Cell. Biochem. 114: 1039–1049, 2013. © 2012 Wiley Periodicals, Inc.     

Adipose-derived stromal/stem cells from different adipose depots in obesity development

The increasing prevalence of obesity is alarming because it is a risk factor for cardiovascular and metabolic diseases(such as type 2 diabetes). The occurrence of these comorbidities in obese patients can arise from white adipose tissue(WAT) dysfunctions, which affect metabolism, insulin sensitivity and promote local and systemic inflammation. In mammals, WAT depots at different anatomical locations(subcutaneous, preperitoneal and visceral) are highly heterogeneous in their morpho-phenotypic profiles and contribute differently to homeostasis and obesity development, depending on their ability to trigger and modulate WAT inflammation. This heterogeneity is likely due to the differential behavior of cells from each depot. Numerous studies suggest that adiposederived stem/stromal cells(ASC; referred to as adipose progenitor cells, in vivo)with depot-specific gene expression profiles and adipogenic and immunomodulatory potentials are keys for the establishment of the morphofunctional heterogeneity between WAT depots, as well as for the development of depot-specific responses to metabolic challenges. In this review, we discuss depot-specific ASC properties and how they can contribute to the pathophysiology of obesity and metabolic disorders, to provide guidance for researchers and clinicians in the development of ASC-based therapeutic approaches.     

Distinct populations of adipogenic and myogenic Myf5-lineage progenitors in white adipose tissues

Brown adipose tissues (BAT) are derived from a myogenic factor 5 (Myf5)-expressing cell lineage and white adipose tissues (WAT) predominantly arise from non-Myf5 lineages, although a subpopulation of adipocytes in some WAT depots can be derived from the Myf5 lineage. However, the functional implication of the Myf5- and non-Myf5-lineage cells in WAT is unclear. We found that the Myf5-lineage constitution in subcutaneous WAT depots is negatively correlated to the expression of classical BAT and newly defined beige/brite adipocyte-specific genes. Consistently, fluorescent-activated cell sorting (FACS)-purified Myf5-lineage adipo-progenitors give rise to adipocytes expressing lower levels of BAT-specific Ucp1, Prdm16, Cidea, and Ppargc1a genes and beige adipocyte-specific CD137, Tmem26, and Tbx1 genes compared with the non-Myf5-lineage adipocytes from the same depots. Ablation of the Myf5-lineage progenitors in WAT stromal vascular cell (SVC) cultures leads to increased expression of BAT and beige cell signature genes. Strikingly, the Myf5-lineage cells in WAT are heterogeneous and contain distinct adipogenic [stem cell antigen 1(Sca1)-positive] and myogenic (Sca1-negative) progenitors. The latter differentiate robustly into myofibers in vitro and in vivo, and they restore dystrophin expression after transplantation into mdx mouse, a model for Duchenne muscular dystrophy. These results demonstrate the heterogeneity and functional differences of the Myf5- and non-Myf5-lineage cells in the white adipose tissue.     

Culture on Tissue‐Specific Coatings Derived from α‐Amylase‐Digested Decellularized Adipose Tissue Enhances the Proliferation and Adipogenic Differentiation of Human Adipose‐Derived Stromal Cells

While extracellular matrix (ECM)‐derived coatings have the potential to direct the response of cell populations in culture, there is a need to investigate the effects of ECM sourcing and processing on substrate bioactivity. To develop improved cell culture models for studying adipogenesis, the current study examines the proliferation and adipogenic differentiation of human adipose‐derived stem/stromal cells (ASCs) on a range of ECM‐derived coatings. Human decellularized adipose tissue (DAT) and commercially available bovine tendon collagen (COL) are digested with α‐amylase or pepsin to prepare the coatings. Physical characterization demonstrates that α‐amylase digestion generates softer, thicker, and more stable coatings, with a fibrous tissue‐like ultrastructure that is lost in the pepsin‐digested thin films. ASCs cultured on the α‐amylase‐digested ECM have a more spindle‐shaped morphology, and proliferation is significantly enhanced on the α‐amylase‐digested DAT coatings. Further, the α‐amylase‐digested DAT provides a more pro‐adipogenic microenvironment, based on higher levels of adipogenic gene expression, glycerol‐3‐phosphate dehydrogenase (GPDH) enzyme activity, and perilipin staining. Overall, this study supports α‐amylase digestion as a new approach for generating bioactive ECM‐derived coatings, and demonstrates tissue‐specific bioactivity using adipose‐derived ECM to enhance ASC proliferation and adipogenic differentiation.     

Stromal stem cells from adipose tissue and bone marrow of age‐matched female donors display distinct immunophenotypic profiles

Adipose tissue is composed of lipid‐filled mature adipocytes and a heterogeneous stromal vascular fraction (SVF) population of cells. Similarly, the bone marrow (BM) is composed of multiple cell types including adipocytes, hematopoietic, osteoprogenitor, and stromal cells necessary to support hematopoiesis. Both adipose and BM contain a population of mesenchymal stromal/stem cells with the potential to differentiate into multiple lineages, including adipogenic, chondrogenic, and osteogenic cells, depending on the culture conditions. In this study we have shown that human adipose‐derived stem cells (ASCs) and bone marrow mesenchymal stem cells (BMSCs) populations display a common expression profile for many surface antigens, including CD29, CD49c, CD147, CD166, and HLA‐abc. Nevertheless, significant differences were noted in the expression of CD34 and its related protein, PODXL, CD36, CD 49f, CD106, and CD146. Furthermore, ASCs displayed more pronounced adipogenic differentiation capability relative to BMSC based on Oil Red staining (7‐fold vs. 2.85‐fold induction). In contrast, no difference between the stem cell types was detected for osteogenic differentiation based on Alizarin Red staining. Analysis by RT‐PCR demonstrated that both the ASC and BMSC differentiated adipocytes and osteoblast displayed a significant upregulation of lineage‐specific mRNAs relative to the undifferentiated cell populations; no significant differences in fold mRNA induction was noted between ASCs and BMSCs. In conclusion, these results demonstrate human ASCs and BMSCs display distinct immunophenotypes based on surface positivity and expression intensity as well as differences in adipogenic differentiation. The findings support the use of both human ASCs and BMSCs for clinical regenerative medicine. J. Cell. Physiol. 226: 843–851, 2011. © 2010 Wiley‐Liss, Inc.     

Differences in Gene Expression and Cytokine Release Profiles Highlight the Heterogeneity of Distinct Subsets of Adipose Tissue-Derived Stem Cells in the Subcutaneous and Visceral Adipose Tissue in Humans

Differences in the inherent properties of adipose tissue-derived stem cells (ASC) may contribute to the biological specificity of the subcutaneous (Sc) and visceral (V) adipose tissue depots. In this study, three distinct subpopulations of ASC, i.e. ASC_SVF, ASC_Bottom, and ASC_Ceiling, were isolated from Sc and V fat biopsies of non-obese subjects, and their gene expression and functional characteristics were investigated. Genome-wide mRNA expression profiles of ASC_SVF, ASC_Bottom and ASC_Ceiling from Sc fat were significantly different as compared to their homologous subsets of V-ASCs. Furthermore, ASC_SVF, ASC_Ceiling and ASC_Bottom from the same fat depot were also distinct from each other. In this respect, both principal component analysis and hierarchical clusters analysis showed that ASC_Ceiling and ASC_SVF shared a similar pattern of closely related genes, which was highly different when compared to that of ASC_Bottom. However, larger variations in gene expression were found in inter-depot than in intra-depot comparisons. The analysis of connectivity of genes differently expressed in each ASC subset demonstrated that, although there was some overlap, there was also a clear distinction between each Sc-ASC and their corresponding V-ASC subsets, and among ASC_SVF, ASC_Bottom, and ASC_Ceiling of Sc or V fat depots in regard to networks associated with regulation of cell cycle, cell organization and development, inflammation and metabolic responses. Finally, the release of several cytokines and growth factors in the ASC cultured medium also showed both inter- and intra-depot differences. Thus, ASC_Ceiling and ASC_Bottom can be identified as two genetically and functionally heterogeneous ASC populations in addition to the ASC_SVF, with ASC_Bottom showing the highest degree of unmatched gene expression. On the other hand, inter-depot seem to prevail over intra-depot differences in the ASC gene expression assets and network functions, contributing to the high degree of specificity of Sc and V adipose tissue in humans.     

Human Ng2+ adipose stem cells loaded in vivo on a new crosslinked hyaluronic acid‐lys scaffold fabricate a skeletal muscle tissue

Mesenchymal stem cell (MSC) therapy holds promise for treating diseases and tissue repair. Regeneration of skeletal muscle tissue that is lost during pathological muscle degeneration or after injuries is sustained by the production of new myofibers. Human Adipose stem cells (ASCs) have been reported to regenerate muscle fibers and reconstitute the pericytic cell pool after myogenic differentiation in vitro. Our aim was to evaluate the differentiation potential of constructs made from a new cross‐linked hyaluronic acid (XHA) scaffold on which different sorted subpopulations of ASCs were loaded. Thirty days after engraftment in mice, we found that NG2⁺ ASCs underwent a complete myogenic differentiation, fabricating a human skeletal muscle tissue, while NG2^? ASCs merely formed a human adipose tissue. Myogenic differentiation was confirmed by the expression of MyoD, MF20, laminin, and lamin A/C by immunofluorescence and/or RT‐PCR. In contrast, adipose differentiation was confirmed by the expression of adiponectin, Glut‐4, and PPAR‐γ. Both tissues formed expressed Class I HLA, confirming their human origin and excluding any contamination by murine cells. In conclusion, our study provides novel evidence that NG2⁺ ASCs loaded on XHA scaffolds are able to fabricate a human skeletal muscle tissue in vivo without the need of a myogenic pre‐differentiation step in vitro. We emphasize the translational significance of our findings for human skeletal muscle regeneration. J. Cell. Physiol. 228: 1762–1773, 2013. © 2013 Wiley Periodicals, Inc.     

In vitro extracorporeal shock wave treatment enhances stemness and preserves multipotency of rat and human adipose-derived stem cells

Background aimsAdipose-derived progenitor/stem cells (ASCs) are discussed as a promising candidate for various tissue engineering approaches. However, its applicability for the clinic is still difficult due to intra- and inter-donor heterogeneity and limited life span in vitro, influencing differentiation capacity as a consequence to decreased multipotency.MethodsExtracorporeal shock wave treatment has been proven to be a suitable clinical tool to improve regeneration of a variety of tissues for several decades, whereas the mechanisms underlying these beneficial effects remain widely unknown.ResultsIn this study we show that human and rat adipose derived stem cells respond strongly to repetitive shock wave treatment in vitro, resulting not only in maintenance and significant elevation of mesenchymal markers (CD73, CD90, CD105), but also in significantly increased differentiation capacity towards the osteogenic and adipogenic lineage as well as toward Schwann-cell like cells even after extended time in vitro, preserving multipotency of ASCs.ConclusionsESWT might be a promising tool to improve ASC quality for cell therapy in various tissue engineering and regenerative medicine applications.     

Character comparison of abdomen‐derived and eyelid‐derived mesenchymal stem cells

Objectives

While most human adipose tissues, such as those located in the abdomen, hip and thigh, are of mesodermal origin, adipose tissues located in the face are of ectodermal origin. The present study has compared stem cell‐related features of abdomen‐derived adult stem cells (A‐ASCs) with those of eyelid‐derived adult stem cells (E‐ASCs).

Materials and methods

Adipose tissue‐derived cells were maintained in DMEM supplemented with 10% FBS. Before passage 6, cells were analysed using FACS, immunocytochemistry and quantitative real time PCR (qRT‐PCR). To examine multi‐differentiational potential, early passage ASCs were cultivated in each of a commercial Stempro^® Differentiation kit.

Results

Unlike fibroblast‐like morphology of A‐ASCs, E‐ASCs had bipolar morphology. Both types of cell exhibited similar surface antigens, and neuronal cell‐related genes and proteins. However, there were differences in mRNA expression levels of CD90 and CD146; neuron‐specific enolase (NSE) and nuclear receptor‐related protein 1 (Nurr1) were different between the two cell types. There was no difference in multi‐differentiational potential between 3 E‐ASCs lines, however, E‐ASCs had higher expression levels of chondrocyte‐related genes compared to A‐ASCs. These cells underwent senescence and maintained normal karyotypes.

Conclusions

Although isolated from similar adipose tissues, both types of cells displayed many contrasting characteristics. Understanding defining phenotypes of such cells is useful for making suitable choices in differing clinical indications.

     

Low‐intensity pulsed ultrasound induced enhanced adipogenesis of adipose‐derived stem cells

Objective

The aim of this study was to investigate effects of low‐intensity pulsed ultrasound (LIPUS) on differentiation of adipose‐derived stem cells (ASCs), in vitro.

Materials and methods

Murine ASCs were treated with LIPUS for either three or five days, immediately after adipogenic induction, or delayed for 2 days. Expression of adipogenic genes PPAR‐γ1, and APN, was examined by real‐time PCR. Immunofluorescence (IF) staining was performed to test for PPAR‐γ at the protein level.

Results

Our data revealed that specific patterns of LIPUS up‐regulated levels of both PPAR‐γ1 and APN mRNA, and PPAR‐γ protein.

Conclusions

In culture medium containing adipogenic reagents, LIPUS enhanced ASC adipogenesis.

     

Spheroids from adipose‐derived stem cells exhibit an miRNA profile of highly undifferentiated cells

Two‐dimensional (2D) cell cultures have been extensively used to investigate stem cell biology, but new insights show that the 2D model may not properly represent the potential of the tissue of origin. Conversely, three‐dimensional cultures exhibit protein expression patterns and intercellular junctions that are more representative of their in vivo condition. Multiclonal cells that grow in suspension are defined as “spheroids,” and we have previously demonstrated that spheroids from adipose‐derived stem cells (S‐ASCs) displayed enhanced regenerative capability. With the current study, we further characterized S‐ASCs to further understand the molecular mechanisms underlying their stemness properties. Recent studies have shown that microRNAs (miRNAs) are involved in many cellular mechanisms, including stemness maintenance and proliferation, and adipose stem cell differentiation. Most studies have been conducted to identify a specific miRNA profile on adherent adipose stem cells, although little is still known about S‐ASCs. In this study, we investigate for the first time the miRNA expression pattern in S‐ASCs compared to that of ASCs, demonstrating that cell lines cultured in suspension show a typical miRNA expression profile that is closer to the one reported in induced pluripotent stem cells. Moreover, we have analyzed miRNAs that are specifically involved in two distinct moments of each differentiation, namely early and late stages of osteogenic, adipogenic, and chondrogenic lineages during long‐term in vitro culture. The data reported in the current study suggest that S‐ASCs have superior stemness features than the ASCs and they represent the true upstream stem cell fraction present in adipose tissue, relegating their adherent counterparts.     

CXCR4‐SF1 bifunctional adipose‐derived stem cells benefit for the treatment of Leydig cell dysfunction‐related diseases

Stem cell transplantation is a candidate method for the treatment of Leydig cell dysfunction‐related diseases. However, there are still many problems that limit its clinical application. Here, we report the establishment of CXCR4‐SF1 bifunctional adipose‐derived stem cells (CXCR4‐SF1‐ADSCs) and their reparative effect on Leydig cell dysfunction. CD29⁺ CD44⁺ CD34^? CD45^? ADSCs were isolated from adipose tissue and purified by fluorescence‐activated cell sorting (FACS). Infection with lentiviruses carrying the CXCR4 and SF1 genes was applied to construct CXCR4‐SF1‐ADSCs. The CXCR4‐SF1‐ADSCs exhibited enhanced migration and had the ability to differentiate into Leydig‐like cells in vitro. Furthermore, the bifunctional ADSCs were injected into BPA‐mediated Leydig cell damage model mice via the tail vein. We found that the CXCR4‐SF1‐ADSCs were capable of homing to the injured testes, differentiating into Leydig‐like cells and repairing the deficiency in reproductive function caused by Leydig cell dysfunction. Moreover, we investigated the mechanism underlying SF1‐mediated differentiation and testosterone synthesis in Leydig cells, and the B‐box and SPRY Domain Containing Protein (BSPRY) gene was proposed to be involved in this process. This study provides insight into the treatment of Leydig cell dysfunction‐related diseases.