Reprogramming Human Adult Fibroblasts into GABAergic Interneurons

Direct reprogramming is an appealing strategy to generate neurons from a somatic cell by forced expression of transcription factors. The generated neurons can be used for both cell replacement strategies and disease modelling. Using this technique, previous studies have shown that γ-aminobutyric acid (GABA) expressing interneurons can be generated from different cell sources, such as glia cells or fetal fibroblasts. Nevertheless, the generation of neurons from adult human fibroblasts, an easily accessible cell source to obtain patient-derived neurons, has proved to be challenging due to the intrinsic blockade of neuronal commitment. In this paper, we used an optimized protocol for adult skin fibroblast reprogramming based on RE1 Silencing Transcription Factor (REST) inhibition together with a combination of GABAergic fate determinants to convert human adult skin fibroblasts into GABAergic neurons. Our results show a successful conversion in 25 days with upregulation of neuronal gene and protein expression levels. Moreover, we identified specific gene combinations that converted fibroblasts into neurons of a GABAergic interneuronal fate. Despite the well-known difficulty in converting adult fibroblasts into functional neurons in vitro, we could detect functional maturation in the induced neurons. GABAergic interneurons have relevance for cognitive impairments and brain disorders, such as Alzheimer’s and Parkinson’s diseases, epilepsy, schizophrenia and autism spectrum disorders.

The effect of mesenchymal stromal cells of various origins on mortality and neurologic deficit in acute ischemia-reperfusion in rats

Stroke is a global epidemic issue and the second leading cause of death in the world and in Ukraine. According to official statistics, every year 100-110 thousand Ukrainians suffer acute cerebrovascular disorders. One third of such patients are of working age, up to 50 % will have a disability, and only one in ten will fully return to full life. So far, promising experimental data on the treatment of neurological dysfunction using mesenchymal stromal cells (MSCs) have been obtained. The aim of study is to compare the effect of MSCs of different origins on mortality and neurologic deficit in rats with acute cerebral ischemia-reperfusion injury (CIRI). Materials and methods. Transient bilateral 20-minute occlusion of internal carotid arteries was modeled in male Wistar rats aged 4 months and animals were injected intravenously with MSCs derived from human umbilical cord Wharton's-jelly (hWJ-MSC), human and rat adipose tissue. Other groups of experimental animals were injected intravenously with rat fetal fibroblasts and cell lysate from hWJ-MSC. The last group of rats received Citicoline at a dose of 250 mg/kg as a reference drug. Control animals were injected intravenously with normal saline. The cerebroprotective effect of therapy was assessed by mortality and neurologic deficit in rats on the McGraw's stroke index score. Results. After 12 hours of observation in the crucial period in the development of experimental acute cerebrovascular disorders with the administration of hWJ-MSC, mortality was only 10 % against 45 % of animals in the control group. The use of rat fetal fibroblasts reduced the mortality of animals compare to the control group by an average of 25 %. CIRI in rats caused severe neurologic deficits: paralysis, paresis, ptosis, circling behavior. On the 7th day of observation in the control group of animals, the mean score on the McGrow's stroke index indicated severe neurological disorders. On the 14th day of observation in this group of animals there was no complete recovery of lost central nervous system functions. Compared with the control group of animals, all the treatment agents for acute CIRI (MSCs of various origins, MSC's lysate and Citicoline) contributed to a significant regression of neurologic deficit. Conclusions. Thus, transplantation of human Wharton's jelly-derived MSCs and rat fetal fibroblasts reduced mortality and alleviated neurological symptoms in rats with experimental ischemic stroke. hWJ-MSC, rat fetal fibroblasts, and rat adipose-derived MSCs reduced the incidence of neurological disorders better than Citicoline, which was accompanied by a regression of neurologic deficit dynamics on the 14th day of follow-up. The ability of stem cells of different origins to reduce neurologic deficit indicates the feasibility of their use in experimental acute cerebral ischemia.

Abnormal Epigenetic Modifications in Surviving Somatic Cell Cloned Cattle Associated With Donor Cell Type

Background Studies have shown that the efficiency of somatic cell nuclear transfer (SCNT) is related to the type of donor cell used. Previous studies have shown that fetal oviduct epithelial cells (FOVs) exhibit a higher blastocyst formation rate than fetal fibroblasts (FFBs), but they are associated with lower pregnancy, calving, and full-term rates after implantation. The reason for this difference is unclear. Result In this study, we performed the assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq), RNA-seq, and 5-hydroxymethylcytidine (5hmC) and 5-methylcytosine (5mC) DNA methylation sequencing methods across the whole genome to analyze the epigenetic differences between cattle cloned using FFBs or FOVs as donor nuclei. The results showed that chromatin openness, gene expression levels, and 5hmC contents were greater in cloned cattle derived from FOV donor nuclei than in those derived from FFBs. ATAC-seq and RNA-seq analyses of cloned bovine ear tissues derived from the same source of donor nuclei showed an obvious clustering tendency. In this study, we also found that the 5hmC content of surviving cloned cattle derived from FFBs was greater than 4‰, whereas it was less than 2‰ in dead cloned cattle. Conclusion We found that there were abnormalities in specific epigenetic modifications and gene expression in living somatic cell cloned cattle derived from different donor nuclei. Although cloned cattle undergo somatic reprogramming and differentiation, they retain the epigenetic imprints of their donor nuclei, and this somatic imprinting may affect the development rate of cloned blastocysts as well as the birth rate and development status of cloned fetuses after implantation.

XLOC-040580, a Novel lncRNA, Modulates Porcine Zygote Activation Progress

Background: Long noncoding RNAs (lncRNAs) play an important role in porcine preimplantation embryonic development, but the regulatory mechanism of lncRNAs during zygote activation (ZGA) remains unclear.Methods: We analyzed the transcriptome data of porcine fetal fibroblasts (PEF), porcine induced pluripotent stem cells (piPSCs) and embryos at different developmental stages before implantation, and found that lncRNAs specifically expressed at ZGA stage including XLOC-040580, and further predicted its target genes TPRA1 and BCL2L1. XLOC-040580 was knocked down by microinjection and the expression of ZGA related genes was detected by qRT-PCR. Microinjection of siRNA corresponding to target genes at the 1-cells, the blastocyst development rate was counted, and single cell sequencing was performed to analyze the regulatory network of target genes and its related mechanisms.Results: By analyzing the transcriptome data of porcine preimplantation embryos, we found that XLOC-040580 was specifically expressed during zygote activation. Knockdown of XLOC-040580 resulted in decreased blastocyst development rate, total blastocyst cell number and TE cell number. XLOC-040580 target genes TPRA1 and BCL2L1 are also specifically expressed in ZGA stage. The knockdown of XLOC-040580 target genes also blocked porcine embryonic development and affected the gene expression of ZGA-related pathways such as cell cycle, histone modification and trophoblast formation during porcine embryonic development.Conclusion: We found and confirmed that lncRNA XLOC-040580 played a key role in the ZGA process, and its target genes (TPRA1, BCL2L1) also coordinated early embryonic development.

Disrupting porcine glutaminase does not block preimplantation development and elongation nor decrease mTORC1 activation in conceptuses

Elongation of pig conceptuses is a dynamic process, requiring adequate nutrient provisions. Glutamine is used as an energy substrate and is involved in the activation of mechanistic target of rapamycin complex 1 (mTORC1) during porcine preimplantation development. However, the roles of glutamine have not been extensively studied past the blastocyst stage. Therefore, the objective of the current study was to determine if glutaminase (GLS), which is the rate-limiting enzyme in glutamine metabolism, was necessary for conceptus elongation to proceed and was involved in mTORC1 activation. The CRISPR/Cas9 system was used to induce loss-of-function mutations in the GLS gene of porcine fetal fibroblasts. Wild type (GLS+/+) and knockout (GLS−/−) fibroblasts were used as donor cells for somatic cell nuclear transfer, and GLS+/+ and GLS−/− blastocyst-stage embryos were transferred into surrogates. On day 14 of gestation, GLS+/+ conceptuses primarily demonstrated filamentous morphologies, and GLS−/− conceptuses exhibited spherical, ovoid, tubular, and filamentous morphologies. Thus, GLS−/− embryos were able to elongate despite the absence of GLS protein and minimal enzyme activity. Furthermore, spherical GLS−/− conceptuses had increased abundance of transcripts related to glutamine and glutamate metabolism and transport compared to filamentous conceptuses of either genotype. Differences in phosphorylation of mTORC1 components and targets were not detected regarding conceptus genotype nor morphology, but abundance of two transcriptional targets of mTORC1, cyclin D1 and peroxisome proliferator-activated receptor gamma coactivator 1-alpha, was increased in spherical conceptuses. Therefore, porcine GLS is not essential for conceptus elongation and is not required for mTORC1 activation at this developmental timepoint.

L-myc Gene Expression in Canine Fetal Fibroblasts Promotes Self-Renewal Capacity but Not Tumor Formation

Canines are useful in mammalian preclinical studies because they are larger than rodents and share many diseases with humans. Canine fetal fibroblast cells (CFFs) are an easily accessible source of somatic cells. However, they are easily driven to senescence and become unusable with continuous in vitro culture. Therefore, to overcome these deficiencies, we investigated whether tetracycline-inducible L-myc gene expression promotes self-renewal activity and tumorigenicity in the production of induced conditional self-renewing fibroblast cells (iCSFCs). Here, we describe the characterization of a new iCSFC line immortalized by transduction with L-myc that displays in vitro self-renewal ability without tumorigenic capacity. We established conditionally inducible self-renewing fibroblast cells by transducing CFF-3 cells with L-myc under the tetracycline-inducible gene expression system. In the absence of doxycycline, the cells did not express L-myc or undergo self-renewal. The iCSFCs had a fibroblast-like morphology, normal chromosome pattern, and expressed fibroblast-specific genes and markers. However, the iCSFCs did not form tumors in a soft agar colony-forming assay. We observed higher expression of three ES modules (core pluripotency genes, polycomb repressive complex genes (PRC), and MYC-related genes) in the iCSFCs than in the CFF-3 cells; in particular, the core pluripotency genes (OCT4, SOX2, and NANOG) were markedly up-regulated compared with the PRC and MYC module genes. These results demonstrated that, in canine fetal fibroblasts, L-myc tetracycline-inducible promoter-driven gene expression induces self-renewal capacity but not tumor formation. This study suggests that L-myc gene-induced conditional self-renewing fibroblast cells can be used as an in vitro tool in a variety of biomedical studies related to drug screening.

Phosphoproteomics Analysis Reveals a Pivotal Mechanism Related to Amino Acid Signals in Goat Fetal Fibroblast

In addition to serving as the building blocks for protein synthesis, amino acids serve as critical signaling molecules in cells. However, the mechanism through which amino acid signals are sensed in cells is not yet fully understood. This study examined differences in the phosphorylation levels of proteins in response to amino acid signals in Cashmere goat fetal fibroblasts (GFb). Amino acid deficiency was found to induce autophagy and attenuate mammalian/mechanistic target of rapamycin complex (mTORC1)/Unc-51-like autophagy activating kinase 1 (ULK1) signaling in GFb cells. A total of 144 phosphosites on 102 proteins positively associated with amino acid signaling were screened using phosphorylation-based proteomics analysis. The mitogen-activated protein kinase (MAPK) signaling pathway was found to play a potentially important role in the interaction network involved in the response to amino acid signals, according to gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis, and MAPK1/3 may serve as a central hub for the entire network. Motif analysis identified three master motifs, xxx_S_Pxx, xxx_S_xxE, and xxx_S_xDx, which were centered on those phosphosites at which phosphorylation was positively regulated by amino acid signaling. Additionally, the phosphorylation levels of three membrane proteins, the zinc transporter SLC39A7, the sodium-dependent neutral amino acid transporters SLC1A5 and SLC38A7, and three translation initiation factors, eukaryotic initiation factor (eIF)5B, eIF4G, and eIF3C, were positively regulated by amino acid signals. These pivotal proteins were added to currently known signaling pathways to generate a novel model of the network pathways associated with amino acid signals. Finally, the phosphorylation levels of threonine 203 and tyrosine 205 on MAPK3 in response to amino acid signals were examined by western blot analysis, and the results were consistent with the data from the phosphoproteomics analysis. The findings of this study provide new evidence and insights into the precise mechanism through which amino acid signals are sensed and conducted in Cashmere goat fetal fibroblasts.

Exogenous LIN28 Is Required for the Maintenance of Self-Renewal and Pluripotency in Presumptive Porcine-Induced Pluripotent Stem Cells

Porcine species have been used in preclinical transplantation models for assessing the efficiency and safety of transplants before their application in human trials. Porcine-induced pluripotent stem cells (piPSCs) are traditionally established using four transcription factors (4TF): OCT4, SOX2, KLF4, and C-MYC. However, the inefficiencies in the reprogramming of piPSCs and the maintenance of their self-renewal and pluripotency remain challenges to be resolved. LIN28 was demonstrated to play a vital role in the induction of pluripotency in humans. To investigate whether this factor is similarly required by piPSCs, the effects of adding LIN28 to the 4TF induction method (5F approach) on the efficiency of piPSC reprogramming and maintenance of self-renewal and pluripotency were examined. Using a retroviral vector, porcine fetal fibroblasts were transfected with human OCT4, SOX2, KLF4, and C-MYC with or without LIN28. The colony morphology and chromosomal stability of these piPSC lines were examined and their pluripotency properties were characterized by investigating both their expression of pluripotency-associated genes and proteins and in vitro and in vivo differentiation capabilities. Alkaline phosphatase assay revealed the reprogramming efficiencies to be 0.33 and 0.17% for the 4TF and 5TF approaches, respectively, but the maintenance of self-renewal and pluripotency until passage 40 was 6.67 and 100%, respectively. Most of the 4TF-piPSC colonies were flat in shape, showed weak positivity for alkaline phosphatase, and expressed a significantly high level of SSEA-4 protein, except for one cell line (VSMUi001-A) whose properties were similar to those of the 5TF-piPSCs; that is, tightly packed and dome-like in shape, markedly positive for alkaline phosphatase, and expressing endogenous pluripotency genes (pOCT4, pSOX2, pNANOG, and pLIN28), significantly high levels of pluripotent proteins (OCT4, SOX2, NANOG, LIN28, and SSEA-1), and a significantly low level of SSEA-4 protein. VSMUi001-A and all 5F-piPSC lines formed embryoid bodies, underwent spontaneous cardiogenic differentiation with cardiac beating, expressed cardiomyocyte markers, and developed teratomas. In conclusion, in addition to the 4TF, LIN28 is required for the effective induction of piPSCs and the maintenance of their long-term self-renewal and pluripotency toward the development of all germ layers. These piPSCs have the potential applicability for veterinary science.

STAT3 Partly Inhibits Cell Proliferation via Direct Negative Regulation of FST Gene Expression

Follistatin (FST) is a secretory glycoprotein and belongs to the TGF-β superfamily. Previously, we found that two single nucleotide polymorphisms (SNPs) of sheep FST gene were significantly associated with wool quality traits in Chinese Merino sheep (Junken type), indicating that FST is involved in the regulation of hair follicle development and hair trait formation. The transcription regulation of human and mouse FST genes has been widely investigated, and many transcription factors have been identified to regulate FST gene. However, to date, the transcriptional regulation of sheep FST is largely unknown. In the present study, genome walking was used to close the genomic gap upstream of the sheep genomic FST gene and to obtain the FST gene promoter sequence. Transcription factor binding site analysis showed sheep FST promoter region contained a conserved putative binding site for signal transducer and activator of transcription 3 (STAT3), located at nucleotides −423 to −416 relative to the first nucleotide (A, +1) of the initiation codon (ATG) of sheep FST gene. The dual-luciferase reporter assay demonstrated that STAT3 inhibited the FST promoter activity and that the mutation of the putative STAT3 binding site attenuated the inhibitory effect of STAT3 on the FST promoter activity. Additionally, chromatin immunoprecipitation assay (ChIP) exhibited that STAT3 is directly bound to the FST promoter. Cell proliferation assay displayed that FST and STAT3 played opposite roles in cell proliferation. Overexpression of sheep FST significantly promoted the proliferation of sheep fetal fibroblasts (SFFs) and human keratinocyte (HaCaT) cells, and overexpression of sheep STAT3 displayed opposite results, which was accompanied by a significantly reduced expression of FST gene (P < 0.05). Taken together, STAT3 directly negatively regulates sheep FST gene and depresses cell proliferation. Our findings may contribute to understanding molecular mechanisms that underlie hair follicle development and morphogenesis.

Efficient TALEN-mediated gene knockin at the bovine Y chromosome and generation of a sex-reversal bovine

Functional elucidation of bovine Y-chromosome genes requires available genome editing technologies. Meanwhile, it has yet to be proven whether the bovine Sry gene is the main or single factor involved in the development of the male phenotype in bovine. Here, we efficiently knocked out four Y-linked genes (Sry, ZFY, DDX3Y, and EIF2S3Y) in bovine fetal fibroblasts (BFFs) with transcription activator-like effector nucleases (TALENs) individually. Furthermore, we used TALEN-mediated gene knockin at the Sry gene and generated a sex-reversal bovine by somatic cell nuclear transfer (SCNT). The resulting bovine had only one ovary and was sterile. We demonstrate, for the first time, that the Sry gene is an important sex-determining gene in bovine. Our method lays a solid foundation for detecting the biology of the bovine Y chromosome, as it may provide an alternative biological model system for the study of mammalian sex determination, and new methods for the practical application in agricultural, especially for sex predetermination.