Rapid conversion of porcine pluripotent stem cells into macrophages with chemically defined conditions.

A renewable source of porcine macrophages derived from pluripotent stem cells (PSCs) would be a valuable alternative to primary porcine alveolar macrophages (PAMs) in the research of host-pathogen interaction mechanisms. We developed an efficient and rapid protocol, within 11 days, to derive macrophages from porcine PSCs (pPSCs). The pPSC-derived macrophages (pPSCdMs) exhibited molecular and functional characteristics of primary macrophages. The pPSCdMs showed macrophage-specific surface protein expression and macrophage-specific transcription factors, similar to PAMs. The pPSCdMs also exhibited the functional characteristics of macrophages, such as endocytosis, phagocytosis, porcine respiratory and reproductive syndrome virus infection and the response to lipopolysaccharide stimulation. Furthermore, we performed transcriptome sequencing of the whole differentiation process to track the fate transitions of porcine PSCs involved in the signaling pathway. The activation of transforming growth factor beta signaling was required for the formation of mesoderm and the inhibition of the transforming growth factor beta signaling pathway at the hematopoietic endothelium stage could enhance the fate transformation of hematopoiesis. In summary, we developed an efficient and rapid protocol to generate pPSCdMs that showed aspects of functional maturity comparable with PAMs. pPSCdMs could provide a broad prospect for the platforms of host-pathogen interaction mechanisms.

Induction of lung progenitor cell-like organoids by porcine pluripotent stem cells.

Organoids are in vitro 3D models that are generated using stem cells to study organ development and regeneration. Despite the extensive research on lung organoids, there is limited information on pig lung cell generation or development. Here, we identified five epithelial cell types along with their characteristic markers using scRNA-seq. Additionally, we found that NKX2.1 and FOXA2 acted as the crucial core transcription factors in porcine lung development. The presence of SOX9/SOX2 double-positive cells was identified as a key marker for lung progenitor cells. The Monocle algorithm was used to create a pseudo-temporal differentiation trajectory of epithelial cells, leading to the identification of signaling pathways related to porcine lung development. Moreover, we established the differentiation method from porcine pluripotent stem cells (pPSCs) to SOX17+ FOXA2+ definitive endoderm (DE) and NKX2.1+ FOXA2+ CDX2- anterior foregut endoderm (AFE). The AFE is further differentiated into lung organoids while closely monitoring the differentiation process. We showed that NKX2.1 overexpression facilitated the induction of lung organoids and supported subsequent lung differentiation and maturation. This model offers valuable insights into studying the interaction patterns between cells and the signaling pathways during the development of the porcine lung.

Super-enhancers conserved within placental mammals maintain stem cell pluripotency.

Despite pluripotent stem cells sharing key transcription factors, their maintenance involves distinct genetic inputs. Emerging evidence suggests that super-enhancers (SEs) can function as master regulatory hubs to control cell identity and pluripotency in humans and mice. However, whether pluripotency-associated SEs share an evolutionary origin in mammals remains elusive. Here, we performed comprehensive comparative epigenomic and transcription factor binding analyses among pigs, humans, and mice to identify pluripotency-associated SEs. Like typical enhancers, SEs displayed rapid evolution in mammals. We showed that BRD4 is an essential and conserved activator for mammalian pluripotency-associated SEs. Comparative motif enrichment analysis revealed 30 shared transcription factor binding motifs among the three species. The majority of transcriptional factors that bind to identified motifs are known regulators associated with pluripotency. Further, we discovered three pluripotency-associated SEs (SE-SOX2, SE-PIM1, and SE-FGFR1) that displayed remarkable conservation in placental mammals and were sufficient to drive reporter gene expression in a pluripotency-dependent manner. Disruption of these conserved SEs through the CRISPR-Cas9 approach severely impaired stem cell pluripotency. Our study provides insights into the understanding of conserved regulatory mechanisms underlying the maintenance of pluripotency as well as species-specific modulation of the pluripotency-associated regulatory networks in mammals.

UCHL1 maintains microenvironmental homeostasis in goat germline stem cells.

Spermatogonial stem cells (SSCs) play a crucial role in mammalian spermatogenesis and maintain the stable inheritance of the germline in livestock. However, stress and bacterial or viral infections can disrupt immune homeostasis of the testes, thereby leading to spermatogenesis destruction and infertility, which severely affects the health and productivity of mammals. This study aimed to explore the effect of ubiquitin C-terminal hydrolase L1 (UCHL1) knockdown (KD) in goat SSCs and mouse testes and investigate the potential anti-inflammatory function of UCHL1 in a poly(I:C)-induced inflammation model to maintain microenvironmental homeostasis. In vitro, the downregulation of UCHL1 (UCHL1 KD) in goat SSCs increased the expression levels of apoptosis and inflammatory factors and inhibited the self-renewal and proliferation of SSCs. In vivo, the structure of seminiferous tubules and spermatogenic cells was disrupted after UCHL1 KD, and the expression levels of apoptosis- and inflammation-related proteins were significantly upregulated. Furthermore, UCHL1 inhibited the TLR3/TBK1/IRF3 pathway to resist poly(I:C)-induced inflammation in SSCs by antagonizing HSPA8 and thus maintaining SSC autoimmune homeostasis. Most importantly, the results of this study showed that UCHL1 maintained immune homeostasis of SSCs and spermatogenesis. UCHL1 KD not only inhibited the self-renewal and proliferation of goat SSCs and spermatogenesis but was also involved in the inflammatory response of goat SSCs. Additionally, UCHL1 has an antiviral function in SSCs by antagonizing HSPA8, which provides an important basis for exploring the specific mechanisms of UCHL1 in goat spermatogenesis.

Interaction between Mesenchymal Stem Cells and Immune Cells during Bone Injury Repair.

Fractures are the most common large organ trauma in humans. The initial inflammatory response promotes bone healing during the initial post-fracture phase, but chronic and persistent inflammation due to infection or other factors does not contribute to the healing process. The precise mechanisms by which immune cells and their cytokines are regulated in bone healing remain unclear. The use of mesenchymal stem cells (MSCs) for cellular therapy of bone injuries is a novel clinical treatment approach. Bone progenitor MSCs not only differentiate into bone, but also interact with the immune system to promote the healing process. We review in vitro and in vivo studies on the role of the immune system and bone marrow MSCs in bone healing and their interactions. A deeper understanding of this paradigm may provide clues to potential therapeutic targets in the healing process, thereby improving the reliability and safety of clinical applications of MSCs to promote bone healing.

Mesenchymal Stem Cells Pretreated with Collagen Promote Skin Wound-Healing.

The existing treatment modalities for skin injuries mainly include dressings, negative-pressure wound treatment, autologous skin grafting, and high-pressure wound treatment. All of these therapies have limitations such as high time cost, the inability to remove inactivated tissue in a timely manner, surgical debridement, and oxygen toxicity. Mesenchymal stem cells have a unique self-renewal ability and wide differentiation potential, and they are one of the most promising stem cell types in cell therapy and have great application prospects in the field of regenerative medicine. Collagen exerts structural roles by promoting the molecular structure, shape, and mechanical properties of cells, and adding it to cell cultures can also promote cell proliferation and shorten the cell doubling time. The effects of collagen on MSCs were examined using Giemsa staining, EdU staining, and growth curves. Mice were subjected to allogeneic experiments and autologous experiments to reduce individual differences; all animals were separated into four groups. Neonatal skin sections were detected by HE staining, Masson staining, immunohistochemical staining, and immunofluorescence staining. We found that the MSCs pretreated with collagen accelerated the healing of skin wounds in mice and canines by promoting epidermal layer repair, collagen deposition, hair follicle angiogenesis, and an inflammatory response. Collagen promotes the secretion of the chemokines and growth factors associated with skin healing by MSCs, which positively influences skin healing. This study supports the treatment of skin injuries with MSCs cultured in medium with collagen added.

Scd1 Deficiency in Early Embryos Affects Blastocyst ICM Formation through RPs-Mdm2-p53 Pathway.

Embryos contain a large number of lipid droplets, and lipid metabolism is gradually activated during embryonic development to provide energy. However, the regulatory mechanisms remain to be investigated. Stearoyl-CoA desaturase 1 (Scd1) is a fatty acid desaturase gene that is mainly involved in intracellular monounsaturated fatty acid production, which takes part in many physiological processes. Analysis of transcripts at key stages of embryo development revealed that Scd1 was important and expressed at an increased level during the cleavage and blastocyst stages. Knockout Scd1 gene by CRISPR/Cas9 from zygotes revealed a decrease in lipid droplets (LDs) and damage in the inner cell mass (ICM) formation of blastocyst. Comparative analysis of normal and knockout embryo transcripts showed a suppression of ribosome protein (RPs) genes, leading to the arrest of ribosome biogenesis at the 2-cell stage. Notably, the P53-related pathway was further activated at the blastocyst stage, which eventually caused embryonic development arrest and apoptosis. In summary, Scd1 helps in providing energy for embryonic development by regulating intra-embryonic lipid droplet formation. Moreover, deficiency activates the RPs-Mdm2-P53 pathway due to ribosomal stress and ultimately leads to embryonic development arrest. The present results suggested that Scd1 gene is essential to maintain healthy development of embryos by regulating energy support.

[Progress on mRNA vaccine for the prevention of major infectious diseases in humans and animals].

A large number of studies have demonstrated that mRNA vaccine has been characterized as a technique with good safety, strong immunogenicity and high developmental potential, which makes it have broad prospects in immunotherapy. In recent years, the stability and in vivo delivery efficiency of mRNA vaccines have been largely addressed by the progresses in mRNA engineering and delivery innovation. And some mRNA vaccines are now clinical approved or in preclinical trials. Here, we summarize current knowledge on the research advances, technology, and application in major infectious diseases in humans and animals of mRNA vaccines, with the aim to provide a reference for improving the development of novel mRNA vaccines.

Comparative analysis of porcine iPSCs derived from Sertoli cells and fibroblasts.

Porcine embryonic fibroblasts (PEFs) can be directly reprogrammed into porcine induced pluripotent stem cells (piPSCs). However, the reprogramming process is generally lengthy and inefficient. Here, we established a fast and efficient induction system of piPSCs from porcine Sertoli cells (SCs) via forced expression of pig Yamanaka factors. The alkaline phosphatase (AP)-positive colonies from SCs developed on Day 3 after lentivirus infection, and were expanded and then picked up on Day 7, whereas reprogramming process from PEFs did not show any colonies in the same period. The picked piPSCs strongly expressed pluripotent genes, had the differentiation capacity to three germ layers, and could be also induced into primordial germ cell-like cells. Screening for transcription factor combinations showed that POU class 5 homeobox 1 (OCT4) is the core factor for AP-positive colony formation, and two factors (OCT4 and c-MYC) could successfully reprogram SCs into piPSCs. We then compared the RNA-sequencing data of piPSCs derived from SCs and PEFs, and found that the most significant difference was the activation of Transforming Growth Factor ß signaling pathway. We also compared the RNA levels of SCs and PEFs, and found that SCs exhibited higher Wnt signaling activity and Bone Morphogenetic Protein 4 expression than PEFs, which might be correlated with higher cell proliferation rate and reprogramming efficiency. In summary, the data demonstrated that starting cell sources of piPSCs significantly affect reprogramming dynamics and SCs could serve as cell sources for efficient reprogramming.

Histone demethylase complexes KDM3A and KDM3B cooperate with OCT4/SOX2 to define a pluripotency gene regulatory network.

The pluripotency gene regulatory network of porcine induced pluripotent stem cells(piPSCs), especially in epigenetics, remains elusive. To determine the biological function of epigenetics, we cultured piPSCs in different culture conditions. We found that activation of pluripotent gene- and pluripotency-related pathways requires the erasure of H3K9 methylation modification which was further influenced by mouse embryonic fibroblast (MEF) served feeder. By dissecting the dynamic change of H3K9 methylation during loss of pluripotency, we demonstrated that the H3K9 demethylases KDM3A and KDM3B regulated global H3K9me2/me3 level and that their co-depletion led to the collapse of the pluripotency gene regulatory network. Immunoprecipitation-mass spectrometry (IP-MS) provided evidence that KDM3A and KDM3B formed a complex to perform H3K9 demethylation. The genome-wide regulation analysis revealed that OCT4 (O) and SOX2 (S), the core pluripotency transcriptional activators, maintained the pluripotent state of piPSCs depending on the H3K9 hypomethylation. Further investigation revealed that O/S cooperating with histone demethylase complex containing KDM3A and KDM3B promoted pluripotency genes expression to maintain the pluripotent state of piPSCs. Together, these data offer a unique insight into the epigenetic pluripotency network of piPSCs.

Peptide-coating 2D and small chemical molecules prolong the passage of porcine spermatogonia stem cells.

Porcine spermatogonia stem cells (pSSCs) are the only type of somatic stem cell that can pass genetic information to the successive generations. Little is known about pSSCs vitality in vitro, and due to their increasing importance in stem cell research, here, we optimized a protocol to culture pSSCs and explored their potential fate in vitro. Utilizing a feeder-free culture system with a 2D peptide-coating and small chemical molecules (including CHIR99021, Repsox, vitamin C, folic acid, and CD lipid concentrate), we were able to prolong the culture time of pSSCs by at least three months compared with previous methods. Moreover, we found that pSSCs could proliferate and self-renew in the seminiferous tubules of infertile mice. However, they could not perform meiosis. Our study shows that this feeder-free culture system optimizes cell culture and may facilitate advanced research on SSC biology and genetic manipulation of germ cells.

DUX: One Transcription Factor Controls 2-Cell-like Fate.

The double homeobox (Dux) gene, encoding a double homeobox transcription factor, is one of the key drivers of totipotency in mice. Recent studies showed Dux was temporally expressed at the 2-cell stage and acted as a transcriptional activator during zygotic genome activation (ZGA) in embryos. A similar activation occurs in mouse embryonic stem cells, giving rise to 2-cell-like cells (2CLCs). Though the molecular mechanism underlying this expanded 2CLC potency caused by Dux activation has been partially revealed, the regulation mechanisms controlling Dux expression remain elusive. Here, we discuss the latest advancements in the multiple levels of regulation of Dux expression, as well as Dux function in 2CLCs transition, aiming to provide a theoretical framework for understanding the mechanisms that regulate totipotency.

Effects of Ferroptosis on Male Reproduction.

Ferroptosis is a relatively novel form of regulated cell death that was discovered in 2012. With the increasing research related to the mechanisms of ferroptosis, previous studies have demonstrated that the inactive of the intracellular antioxidant system and iron overload can result in the accumulation of reactive oxygen species (ROS), which can ultimately cause lipid peroxidation in the various cell types of the body. ROS accumulation can cause sperm damage by attacking the plasma membrane and damaging DNA. Acute ferroptosis causes oxidative damage to sperm DNA and testicular oxidative stress, thereby causing male reproductive dysfunction. This review aims to discuss the metabolic network of ferroptosis, summarize and analyze the relationship between male reproductive diseases caused by iron overload as well as lipid peroxidation, and provide a novel direction for the research and prevention of various male reproductive diseases.

Placenta-Specific miR-125b Overexpression Leads to Increased Rates of Pregnancy Loss in Mice.

Pregnancy loss (PL) is one of the common complications that women can experience during pregnancy, with an occurrence rate of 1 to 5%. The potential causes of pregnancy loss are unclear, with no effective treatment modalities being available. It has been previously reported that the level of miR-125b was significantly increased in placentas of PL patients. However, the role of miR-125b in the development of PL still remains unknown. In the current study, an miR-125b placenta-specific over-expression model was constructed by lentiviral transfecting zona-free mouse embryos followed by embryo transfer. On gestation day 15, it was observed that the placenta was significantly smaller in the miR-125b placenta-specific overexpression group than the control group. Additionally, the abortion rate of the miR-125b placenta-specific overexpression group was markedly higher than in the control group. The blood vessel diameter was larger in the miR-125b-overexpressing specific placenta. In addition, miR-125b-overexpressing HTR8 and JEG3 cell lines were also generated to analyze the migration and invasion ability of trophoblasts. The results showed that miR-125b overexpression significantly suppressed the migration and invasion ability of HTR8 and JEG3 cells. Overall, our results demonstrated that miR-125b can affect embryo implantation through modulating placenta angiogenesis and trophoblast cell invasion capacity that can lead to PL.

KDM4C Contributes to Trophoblast-like Stem Cell Conversion from Porcine-Induced Pluripotent Stem Cells (piPSCs) via Regulating CDX2.

Studies on ESRRB-regulating porcine-induced pluripotent stem cells (piPSCs) converted to trophoblast-like stem cells (TLSCs) contribute to the understanding of early embryo development. However, the epigenetic modification regulation network during the conversion is poorly understood. Here, the global change in histone H3 Lysine 4, 9, 27, 36 methylation and Lysine 27 acetylation was investigated in piPSCs and TLSCs. We found a high modification profile of H3K36me2 in TLSCs compared to that of piPSCs, whereas the profiles of other modifications remained constant. KDM4C, a H3K36me3/2 demethylase, whose gene body region was combined with ESRRB, was upregulated in TLSCs. Moreover, KDM4 inhibitor supplementation rescued the AP-negative phenotype observed in TLSCs, confirming that KDM4C could regulate the pluripotency of TLSCs. Subsequently, KDM4C replenishment results show the significantly repressed proliferation and AP-positive staining of TLSCs. The expressions of CDX2 and KRT8 were also upregulated after KDM4C overexpression. In summary, these results show that KDM4C replaced the function of ESRRB. These findings reveal the unique and crucial role of KDM4C-mediated epigenetic chromatin modifications in determination of piPSCs' fate and expand the understanding of the connection between piPSCs and TSCs.

Melatonin alleviates LPS-induced endoplasmic reticulum stress and inflammation in spermatogonial stem cells.

Orchitis is one of the leading causes of male animal infertility and is associated with inflammatory reactions caused by the bacterium. It has been reported that there is a mutual coupling effect between endoplasmic reticulum stress (ERS) and inflammatory response. Our studies showed that lipopolysaccharide (LPS) could cause testicular damages, apoptosis, ERS, and inflammatory responses in spermatogonial stem cells (SSCs); ERS-related apoptosis proteins were activated and the expression of ERS genes was significantly upregulated; meanwhile, the expression of Toll-like receptor 4 and inflammation factors was apparently increased with LPS treatment. Moreover, melatonin (MEL) could rescue testicular damage, and significantly inhibited the expression of ERS-related apoptosis genes, ERS markers, and inflammatory factors in SSCs and MEL played repairing and anti-infection roles in LPS-induced testicular damage. Therefore, MEL may be used as a drug to prevent and control bacterial infections in male reproductive systems. However, the specific molecular mechanism of MEL to resist ERS and inflammatory response remains to be further studied.

PAX7 promotes CD49f-positive dairy goat spermatogonial stem cells' self-renewal.

Spermatogenesis is a complex process that originates from and depends on the spermatogonial stem cells (SSCs). The number of SSCs is rare, which makes the separation and enrichment of SSCs difficult and inefficient. The transcription factor PAX7 maintains fertility in normal spermatogenesis in mice. However, for large animals, much less is known about the SSCs' self-renewal regulation, especially in dairy goats. We isolated and enriched the CD49f-positive and negative dairy goat testicular cells by magnetic-activated cell sorting strategies. The RNA- sequencing and experimental data revealed that cells with a high CD49f and PAX7 expression are undifferentiated spermatogonia in goat testis. Our findings indicated that ZBTB16 (PLZF), PAX7, LIN28A, BMPR1B, FGFR1, and FOXO1 were expressed higher in CD49f-positive cells as compared to negative cells and goat fibroblasts cells. The expression and distribution of PAX7 in dairy goat also have been detected, which gradually decreased in testis tissue along with the increasing age. When the PAX7 gene was overexpressed in dairy goat immortal mGSCs-I-SB germ cell lines, the expression of PLZF, GFRα1, ID4, and OCT4 was upregulated. Together, our data demonstrated that there is a subset of spermatogonial stem cells with a high expression of PAX7 among the CD49f+ spermatogonia, and PAX7 can maintain the self-renewal of CD49f-positive SSCs.

Interaction between DMRT1 and PLZF protein regulates self-renewal and proliferation in male germline stem cells.

Double sex and mab-3 related transcription factor 1 (DMRT1) encodes a double sex/mab-3 (DM) domain, which is the most conserved structure that involved in sex determination both in vertebrates and invertebrates. This study revealed important roles of DMRT1 in maintaining self-renewal of male germline stem cells (mGSCs). Our results showed that insufficient expression of DMRT1 in mice testes resulted in decreased number of spermatogonial cells and collapse of testicular niche in vivo. Self-renewal and proliferation of mGSCs were inhibited. Based on the bimolecular fluorescence complementation (BiFC) and co-immunoprecipitation (co-IP) assay, it was finally revealed that the interaction between DMRT1 and promyelocytic leukemia zinc finger (PLZF) protein was essential for maintaining self-renewal of mGSCs. Moreover, BTB domain of PLZF, DM and DMRT1 domain of DMRT1 were indispensable in mGSC, which were responsible for preserving the quantity of germ cells. Our research provided a new scientific basis for studying the mechanism of self-renewal and spermatogenesis in goat mGSCs.

X-box binding protein 1 (XBP1) function in diseases.

The accumulation of unfolded or misfolded proteins in the endoplasmic reticulum (ER) causes endoplasmic reticulum stress (ERS), which is characteristic of cells with high levels of secretory activity and is involved in a variety of diseases. In response to ERS, cells initiate an adaptive process named the unfolding protein response (UPR) to maintain intracellular homeostasis and survival. However, long term and unresolved ERS can also induce apoptosis. As the most conserved signaling branch of UPR, the IRE1-XBP1 pathway plays an important role in both physiological and pathological states, and its activity has a profound impact on disease progression and prognosis. Here, the latest research progress of IRE1-XBP1 pathway in cancer, metabolic diseases, and other diseases was briefly introduced, and the relationship between several diseases and this pathway was analyzed. Besides, the new understanding and prospect of IRE1-XBP1 pathway regulating male reproduction were reviewed.

AXIN2 Reduces the Survival of Porcine Induced Pluripotent Stem Cells (piPSCs).

The establishment of porcine pluripotent stem cells (piPSCs) is critical but remains challenging. All piPSCs are extremely sensitive to minor perturbations of culture conditions and signaling network. Inhibitors, such as CHIR99021 and XAV939 targeting the WNT signaling pathway, have been added in a culture medium to modify the cell regulatory network. However, potential side effects of inhibitors could confine the pluripotency and practicability of piPSCs. This study aimed to investigate the roles of AXIN, one component of the WNT pathway in piPSCs. Here, porcine AXIN1 and AXIN2 genes were knocked-down or overexpressed. Digital RNA-seq was performed to explore the mechanism of cell proliferation and apoptosis. We found that (1) overexpression of the porcine AXIN2 gene significantly reduced survival and negatively impacted the pluripotency of piPSCs, and (2) knockdown of AXIN2, a negative effector of the WNT signaling pathway, enhanced the expression of genes involved in cell cycle but reduced the expression of genes related to cell differentiation, death, and apoptosis.