Ultrastructural heterogeneity of the mitochondrial population in rat embryonic and induced pluripotent stem cells.

Even though rats are popular model animals, the ultrastructure of their pluripotent cells, that is, embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), remains unexplored, although fine structure of pluripotent stem cells of mice and humans and its changes during differentiation have been investigated well. In the present study, we carried out ultrastructural and morphometric analyses of three lines of rat ESCs and two lines of rat iPSCs. The rat pluripotent stem cells were found to have the main typical morphological features of pluripotent cells: large nuclei of irregular or nearly round shape, scanty cytoplasm with few membrane organelles, and a poorly developed Golgi apparatus and endoplasmic reticulum. The cytoplasm of the rat pluripotent cells contains clusters of glycogen, previously described in human ESCs. To identify possible differences between rat ESCs and iPSCs, we performed a morphometric analysis of cell parameters. The mean area of cells and nuclei, the nuclear/cytoplasmic ratio, distributions of glycogen and diversity of mitochondria showed marked variations among the lines of rat pluripotent stem cells and were more pronounced than variations between rat ESCs and iPSCs as separate types of pluripotent stem cells. We noted morphological heterogeneity of the mitochondrial population in the rat pluripotent stem cells. The cells contained three types of mitochondria differing in the structure of cristae and in matrix density, and our morphometric analysis revealed differences in cristae structure.

Embryonic Stem Cell-Derived Extracellular Vesicles Maintain ESC Stemness by Activating FAK.

It is critical that epiblast cells within blastocyst-stage embryos receive the necessary regulatory cues to remain pluripotent until the appropriate time when they are stimulated to undergo differentiation, ultimately to give rise to an entire organism. Here, we show that exposure of embryonic stem cells (ESCs), which are the in vitro equivalents of epiblasts, to ESC-derived extracellular vesicles (EVs) helps to maintain their stem cell properties even under culture conditions that would otherwise induce differentiation. EV-treated ESCs continued to express stemness genes, preserving their pluripotency and ability to generate chimeric mice. These effects were triggered by fibronectin bound to the surfaces of EVs, enabling them to interact with ESC-associated integrins and activate FAK more effectively than fibronectin alone. Overall, these findings highlight a potential regulatory mechanism whereby epiblast cells, via their shed EVs, create an environment within the blastocyst that prevents their premature differentiation and maintains their pluripotent state.

Regulation of single-cell genome organization into TADs and chromatin nanodomains.

The genome folds into a hierarchy of three-dimensional structures within the nucleus. At the sub-megabase scale, chromosomes form topologically associating domains (TADs)1-4. However, how TADs fold in single cells is elusive. Here, we reveal TAD features inaccessible to cell population analysis by using super-resolution microscopy. TAD structures and physical insulation associated with their borders are variable between individual cells, yet chromatin intermingling is enriched within TADs compared to adjacent TADs in most cells. The spatial segregation of TADs is further exacerbated during cell differentiation. Favored interactions within TADs are regulated by cohesin and CTCF through distinct mechanisms: cohesin generates chromatin contacts and intermingling while CTCF prevents inter-TAD contacts. Furthermore, TADs are subdivided into discrete nanodomains, which persist in cells depleted of CTCF or cohesin, whereas disruption of nucleosome contacts alters their structural organization. Altogether, these results provide a physical basis for the folding of individual chromosomes at the nanoscale.

Genomic Repeats Categorize Genes with Distinct Functions for Orchestrated Regulation.

Repetitive elements are abundantly distributed in mammalian genomes. Here, we reveal a striking association between repeat subtypes and gene function. SINE, L1, and low-complexity repeats demarcate distinct functional categories of genes and may dictate the time and level of gene expression by providing binding sites for different regulatory proteins. Importantly, imaging and sequencing analysis show that L1 repeats sequester a large set of genes with specialized functions in nucleolus- and lamina-associated inactive domains that are depleted of SINE repeats. In addition, L1 transcripts bind extensively to its DNA in embryonic stem cells (ESCs). Depletion of L1 RNA in ESCs leads to relocation of L1-enriched chromosomal segments from inactive domains to the nuclear interior and de-repression of L1-associated genes. These results demonstrate a role of L1 DNA and RNA in gene silencing and suggest a general theme of genomic repeats in orchestrating the function, regulation, and expression of their host genes.

Use of embryonic stem cell-derived cardiomyocytes to study cardiotoxicity of bisphenol AF via the GPER/CAM/eNOS pathway.

Bisphenol AF (BPAF) is a derivative of bisphenol A (BPA) that is widely used in fluorinated polymers, fluorinated rubber, electronic equipment, plastic optical fibers, etc. Studies have shown that BPAF exposure is associated with a number of diseases; however, little is known about the effects of BPAF on cardiomyocytes. We investigated the impact of chronic exposure to BPAF on cardiomyocytes derived from embryonic stem cells (ESCs). The present study showed that chronic exposure to various concentrations of BPAF (0, 8, 200 and 1000 ng/ml) induces cardiomyocyte hypertrophy. The ratios of microfilaments to mitochondrial length and the ratio of microfilaments to cell nuclei and MYH7b levels indicate that BPAF exposure alters the morphology of the cells and mitochondria. Furthermore, BPAF exposure at concentrations from 8 to 1000 ng/ml results in an increase in G protein-coupled estrogen receptor (GPER) expression. Additionally, our results suggest that these effects of BPAF mediate cardiomyocyte hypertrophy apparently due to an increase in the production of reactive nitrogen species (RNS) via an increase in endothelial NO synthase (eNOS). These results imply that ESC-based myocardial differentiation can be an excellent cellular model to study BPAF-induced cardiotoxicity at the cellular and molecular levels.

SEM/FIB Imaging for Studying Neural Interfaces.

Tissue and neural engineering for various regenerative therapies are rapidly growing fields. Of major interest is studying the complex interface between cells and various 3D structures by scanning electron microscopy with focused ion beam. Notwithstanding its unrivaled resolution, the optimal fixation, dehydration, and staining protocols of the samples while preserving the complex cell interface in its natural form, are highly challenging. The aim of this work was to compare and optimize staining and sample drying procedures in order to preserve the cells in their "life-like state" for studying the cell interface with either 3D well-like structures or gold-coated mushroom-shaped electrodes. The process involved chemical fixation using a combination of glutaraldehyde and formaldehyde, followed by gentle drying techniques in which we compared four methods: (critical point drying, hexamethyldisiloxane, repeats of osmium tetroxide-thiocarbohydrazide [OTOTO], and resin) in order to determine the method that best preserves the cell and cell interface morphology. Finally, to visualize the intracellular organelles and membrane, we compared the efficacy of four staining techniques: osmium tetroxide, osmium tetroxide and salts, osmium and uranyl acetate, and OTOTO. Experiments were performed on embryonic stem cell-derived photoreceptor precursors, neural cells, and a human retinal pigment epithelial cell line, which revealed that the optimal processing combination was resin drying and OTOTO staining, as manifested by preservation of cell morphology, the lowest percentage of cellular protrusion breakage as well as a high-quality image. The obtained results pave the way for better understanding the cell interface with various structures for enhancing various biomedical applications.

Spatiotemporal Characterizations of Spontaneously Beating Cardiomyocytes with Adaptive Reference Digital Image Correlation.

We developed an Adaptive Reference-Digital Image Correlation (AR-DIC) method that enables unbiased and accurate mechanics measurements of moving biological tissue samples. We applied the AR-DIC analysis to a spontaneously beating cardiomyocyte (CM) tissue, and could provide correct quantifications of tissue displacement and strain for the beating CMs utilizing physiologically-relevant, sarcomere displacement length-based contraction criteria. The data were further synthesized into novel spatiotemporal parameters of CM contraction to account for the CM beating homogeneity, synchronicity, and propagation as holistic measures of functional myocardial tissue development. Our AR-DIC analyses may thus provide advanced non-invasive characterization tools for assessing the development of spontaneously contracting CMs, suggesting an applicability in myocardial regenerative medicine.

High glucose concentrations mask cellular phenotypes in a stem cell model of tuberous sclerosis complex.

Tuberous sclerosis complex (TSC) is a neurodevelopmental disorder caused by deletions in the TSC1 or TSC2 genes that is associated with epilepsy in up to 90% of patients. Seizures are suggested to start in benign brain tumors, cortical tubers, or in the perituberal tissue making these tubers an interesting target for further research into mechanisms underlying epileptogenesis in TSC. Animal models of TSC insufficiently capture the neurodevelopmental biology of cortical tubers, and hence, human stem cell-based in vitro models of TSC are being increasingly explored in attempts to recapitulate tuber development and epileptogenesis in TSC. However, in vitro culture conditions for stem cell-derived neurons do not necessarily mimic physiological conditions. For example, very high glucose concentrations of up to 25 mM are common in culture media formulations. As TSC is potentially caused by a disruption of the mechanistic target of rapamycin (mTOR) pathway, a main integrator of metabolic information and intracellular signaling, we aimed to examine the impact of different glucose concentrations in the culture media on cellular phenotypes implicated in tuber characteristics. Here, we present preliminary data from a pilot study exploring cortical neuronal differentiation on human embryonic stem cells (hES) harboring a TSC2 knockout mutation (TSC2-/-) and an isogenic control line (TSC2+/+). We show that the commonly used high glucose media profoundly mask cellular phenotypes in TSC2-/- cultures during neuronal differentiation. These phenotypes only become apparent when differentiating TSC2+/+ and TSC2-/- cultures in more physiologically relevant conditions of 5 mM glucose suggesting that the careful consideration of culture conditions is vital to ensuring biological relevance and translatability of stem cell models for neurological disorders such as TSC. This article is part of the Special Issue "Proceedings of the 7th London-Innsbruck Colloquium on Status Epilepticus and Acute Seizures".

Non-invasive detection of DNA methylation states in carcinoma and pluripotent stem cells using Raman microspectroscopy and imaging.

DNA methylation plays a critical role in the regulation of gene expression. Global DNA methylation changes occur in carcinogenesis as well as early embryonic development. However, the current methods for studying global DNA methylation levels are invasive and require sample preparation. The present study was designed to investigate the potential of Raman microspectroscopy and Raman imaging as non-invasive, marker-independent and non-destructive tools for the detection of DNA methylation in living cells. To investigate global DNA methylation changes, human colon carcinoma HCT116 cells, which were hypomorphic for DNA methyltransferase 1, therefore showing a lower global DNA methylation (DNMT1-/- cells), were compared to HCT116 wildtype cells. As a model system for early embryogenesis, murine embryonic stem cells were adapted to serum-free 2i medium, leading to a significant decrease in DNA methylation. Subsequently, 2i medium -adapted cells were compared to cells cultured in serum-containing medium. Raman microspectroscopy and imaging revealed significant differences between high- and low-methylated cell types. Higher methylated cells demonstrated higher relative intensities of Raman peaks, which can be assigned to the nucleobases and 5-methylcytosine. Principal component analysis detected distinguishable populations of high- and low-methylated samples. Based on the provided data we conclude that Raman microspectroscopy and imaging are suitable tools for the real-time, marker-independent and artefact-free investigation of the DNA methylation states in living cells.

Pancreatic duct-like cell line derived from pig embryonic stem cells: expression of uroplakin genes in pig pancreatic tissue.

The isolation of a cell line, PICM-31D, with phenotypic characteristics like pancreatic duct cells is described. The PICM-31D cell line was derived from the previously described pig embryonic stem cell-derived exocrine pancreatic cell line, PICM-31. The PICM-31D cell line was morphologically distinct from the parental cells in growing as a monolayer rather than self-assembling into multicellular acinar-like structures. The PICM-31D cells were propagated for over a year at split ratios of 1:3 to 1:10 at each passage without change in phenotype or growth rate. Electron microscopy showed the cells to be a polarized epithelium of cuboidal cells joined by tight junction-like adhesions at their apical/lateral aspect. The cells contained numerous mucus-like secretory vesicles under their apical cell membrane. Proteomic analysis of the PICM-31D's cellular proteins detected MUC1 and MUC4, consistent with mucus vesicle morphology. Gene expression analysis showed the cells expressed pancreatic ductal cell-related transcription factors such as GATA4, GATA6, HES1, HNF1A, HNF1B, ONECUT1 (HNF6), PDX1, and SOX9, but little or no pancreas progenitor cell markers such as PTF1A, NKX6-1, SOX2, or NGN3. Pancreas ductal cell-associated genes including CA2, CFTR, MUC1, MUC5B, MUC13, SHH, TFF1, KRT8, and KRT19 were expressed by the PICM-31D cells, but the exocrine pancreas marker genes, CPA1 and PLA2G1B, were not expressed by the cells. However, the exocrine marker, AMY2A, was still expressed by the cells. Surprisingly, uroplakin proteins were prominent in the PICM-31D cell proteome, particularly UPK1A. Annexin A1 and A2 proteins were also relatively abundant in the cells. The expression of the uroplakin and annexin genes was detected in the cells, although only UPK1B, UPK3B, ANXA2, and ANXA4 were detected in fetal pig pancreatic duct tissue. In conclusion, the PICM-31D cell line models the mucus-secreting ductal cells of the fetal pig pancreas.

The Origin of a New Progenitor Stem Cell Group in Human Development.

The observation of two precursor groups of the early stem cells (Groups I and II) leads to the realization that a first amount of fetal stem cells (Group I) migrate from the AMG (Aortal-Mesonephric-Gonadal)-region into the aorta and its branching vessels. A second group (Group II) gains quite a new significance during human development. This group presents a specific developmental step which is found only in the human. This continuation of the early development along a different way indicates a general alteration of the stem cell biology. This changed process in the stem cell scene dominates the further development of the human stem cells. It remains unclear where this phylogenetic step first appears. By far not all advanced mammals show this second group of stem cells and their axonal migration. Essentially only primates seem to be involved in this special development.

NANOG Is Required for the Long-Term Establishment of Avian Somatic Reprogrammed Cells.

Somatic reprogramming, which was first identified in rodents, remains poorly described in non-mammalian species. Here, we generated avian reprogrammed cells by reprogramming of chicken and duck primary embryonic fibroblasts. The efficient generation of long-term proliferating cells depends on the method of delivery of reprogramming factors and the addition of NANOG and LIN28 to the canonical OCT4, SOX2, KLF4, and c-MYC gene combination. The reprogrammed cells were positive for several key pluripotency-associated markers including alkaline phosphatase activity, telomerase activity, SSEA1 expression, and specific cell cycle and epigenetic markers. Upregulated endogenous pluripotency-associated genes included POU5F3 (POUV) and KLF4, whereas cells failed to upregulate NANOG and LIN28A. However, cells showed a tumorigenic propensity when injected into recipient embryos. In conclusion, although the somatic reprogramming process is active in avian primary cells, it needs to be optimized to obtain fully reprogrammed cells with similar properties to those of chicken embryonic stem cells.

Analysis of Mitochondrial Dimensions and Cristae Structure in Pluripotent Stem Cells Using Transmission Electron Microscopy.

Dynamic alterations to mitochondrial structure and function regulate cell fate decisions and underlie multiple age-related and genetic diseases that are modeled using embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs). Transmission electron microscopy (TEM) can be used to obtain high-resolution micrographs of mitochondria, but mitochondrial ultrastructure is easily distorted during specimen processing. This unit describes a method that preserves mitochondrial membrane structure from adherent ESC cultures for TEM sample preparation. This procedure is useful for assessing ultrastructural changes to mitochondria during differentiation, reprogramming, or experimental manipulation of ESC metabolism. We provide comprehensive protocols for: (1) preparation of ESC cultures for TEM; (2) retrieval of thin sections from individual ESCs; and (3) contrast staining and morphometric analysis of mitochondria and cristae. This unit also describes an alternative procedure for samples with low cell numbers and a supporting protocol for morphometric image analysis. Collectively, these protocols allow for the observation and quantitative analysis of mitochondria in ESCs. © 2018 by John Wiley & Sons, Inc.

Acellular therapeutic approach for heart failure: in vitro production of extracellular vesicles from human cardiovascular progenitors.

Aims: We have shown that extracellular vesicles (EVs) secreted by embryonic stem cell-derived cardiovascular progenitor cells (Pg) recapitulate the therapeutic effects of their parent cells in a mouse model of chronic heart failure (CHF). Our objectives are to investigate whether EV released by more readily available cell sources are therapeutic, whether their effectiveness is influenced by the differentiation state of the secreting cell, and through which mechanisms they act. Methods and results: The total EV secreted by human induced pluripotent stem cell-derived cardiovascular progenitors (iPSC-Pg) and human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CM) were isolated by ultracentrifugation and characterized by Nanoparticle Tracking Analysis, western blot, and cryo-electron microscopy. In vitro bioactivity assays were used to evaluate their cellular effects. Cell and EV microRNA (miRNA) content were assessed by miRNA array. Myocardial infarction was induced in 199 nude mice. Three weeks later, mice with left ventricular ejection fraction (LVEF) ≤ 45% received transcutaneous echo-guided injections of iPSC-CM (1.4 × 106, n = 19), iPSC-Pg (1.4 × 106, n = 17), total EV secreted by 1.4 × 106 iPSC-Pg (n = 19), or phosphate-buffered saline (control, n = 17) into the peri-infarct myocardium. Seven weeks later, hearts were evaluated by echocardiography, histology, and gene expression profiling, blinded to treatment group. In vitro, EV were internalized by target cells, increased cell survival, cell proliferation, and endothelial cell migration in a dose-dependent manner and stimulated tube formation. Extracellular vesicles were rich in miRNAs and most of the 16 highly abundant, evolutionarily conserved miRNAs are associated with tissue-repair pathways. In vivo, EV outperformed cell injections, significantly improving cardiac function through decreased left ventricular volumes (left ventricular end systolic volume: -11%, P < 0.001; left ventricular end diastolic volume: -4%, P = 0.002), and increased LVEF (+14%, P < 0.0001) relative to baseline values. Gene profiling revealed that EV-treated hearts were enriched for tissue reparative pathways. Conclusion: Extracellular vesicles secreted by iPSC-Pg are effective in the treatment of CHF, possibly, in part, through their specific miRNA signature and the associated stimulation of distinct cardioprotective pathways. The processing and regulatory advantages of EV could make them effective substitutes for cell transplantation.

Human Astrocytes Transfer Aggregated Alpha-Synuclein via Tunneling Nanotubes.

Many lines of evidence suggest that the Parkinson's disease (PD)-related protein α-synuclein (α-SYN) can propagate from cell to cell in a prion-like manner. However, the cellular mechanisms behind the spreading remain elusive. Here, we show that human astrocytes derived from embryonic stem cells actively transfer aggregated α-SYN to nearby astrocytes via direct contact and tunneling nanotubes (TNTs). Failure in the astrocytes' lysosomal digestion of excess α-SYN oligomers results in α-SYN deposits in the trans-Golgi network followed by endoplasmic reticulum swelling and mitochondrial disturbances. The stressed astrocytes respond by conspicuously sending out TNTs, enabling intercellular transfer of α-SYN to healthy astrocytes, which in return deliver mitochondria, indicating a TNT-mediated rescue mechanism. Using a pharmacological approach to inhibit TNT formation, we abolished the transfer of both α-SYN and mitochondria. Together, our results highlight the role of astrocytes in α-SYN cell-to-cell transfer, identifying possible pathophysiological events in the PD brain that could be of therapeutic relevance.SIGNIFICANCE STATEMENT Astrocytes are the major cell type in the brain, yet their role in Parkinson's disease progression remains elusive. Here, we show that human astrocytes actively transfer aggregated α-synuclein (α-SYN) to healthy astrocytes via direct contact and tunneling nanotubes (TNTs), rather than degrade it. The astrocytes engulf large amounts of oligomeric α-SYN that are subsequently stored in the trans-Golgi network region. The accumulation of α-SYN in the astrocytes affects their lysosomal machinery and induces mitochondrial damage. The stressed astrocytes respond by sending out TNTs, enabling intercellular transfer of α-SYN to healthy astrocytes. Our findings highlight an unexpected role of astrocytes in the propagation of α-SYN pathology via TNTs, revealing astrocytes as a potential target for therapeutic intervention.

Revealing 3D Ultrastructure and Morphology of Stem Cell Spheroids by Electron Microscopy.

Cell culture methods have been developed in efforts to produce biologically relevant systems for developmental and disease modeling, and appropriate analytical tools are essential. Knowledge of ultrastructural characteristics represents the basis to reveal in situ the cellular morphology, cell-cell interactions, organelle distribution, niches in which cells reside, and many more. The traditional method for 3D visualization of ultrastructural components, serial sectioning using transmission electron microscopy (TEM), is very labor-intensive due to contentious TEM slice preparation and subsequent image processing of the whole collection. In this chapter, we present serial block-face scanning electron microscopy, together with complex methodology for spheroid formation, contrasting of cellular compartments, image processing, and 3D visualization. The described technique is effective for detailed morphological analysis of stem cell spheroids, organoids, as well as organotypic cell cultures.

Derivation and Characterization of a Pig Embryonic-Stem-Cell-Derived Exocrine Pancreatic Cell Line.

OBJECTIVES: The aim of this study was to identify an epithelial cell line isolated from the spontaneous differentiation of totipotent pig epiblast cells. METHODS: PICM-31 and its colony-cloned derivative cell line, PICM-31A, were established from the culture and differentiation of an epiblast mass isolated from an 8-day-old pig blastocyst. The cell lines were analyzed by transmission electron microscopy, marker gene expression, and mass spectroscopy-based proteomics. RESULTS: The PICM-31 cell lines were continuously cultured and could be successively colony cloned. They spontaneously self-organized into acinarlike structures. Transmission electron microscopy indicated that the cell lines' cells were epithelial and filled with secretory granules. Candidate gene expression analysis of the cells showed an exocrine pancreatic profile that included digestive enzyme expression, for example, carboxypeptidase A1, and expression of the fetal marker, α-fetoprotein. Pancreatic progenitor marker expression included pancreatic and duodenal homeobox 1, NK6 homeobox 1, and pancreas-specific transcription factor 1a, but not neurogenin 3. Proteomic analysis of cellular proteins confirmed the cells' production of digestive enzymes and showed that the cells expressed cytokeratins 8 and 18. CONCLUSIONS: The PICM-31 cell lines provide in vitro models of fetal pig pancreatic exocrine cells. They are the first demonstration of continuous cultures, that is, cell lines, of nontransformed pig pancreas cells.

Coupling between chromosome intermingling and gene regulation during cellular differentiation.

In this article, we summarize current findings for the emergence of biophysical properties such as nuclear stiffness, chromatin compaction, chromosome positioning, and chromosome intermingling during stem cell differentiation, which eventually correlated with the changes of gene expression profiles during cellular differentiation. An overview is first provided to link stem cell differentiation with alterations in nuclear architecture, chromatin compaction, along with nuclear and chromatin dynamics. Further, we highlight the recent biophysical and molecular approaches, imaging methods and computational developments in characterizing transcription-related chromosome organization especially chromosome intermingling and nano-scale chromosomal contacts. Finally, the article ends with an outlook towards the emergence of a functional roadmap in setting up chromosome positioning and intermingling in a cell type specific manner during cellular differentiation.

DNA methylation is dispensable for changes in global chromatin architecture but required for chromocentre formation in early stem cell differentiation.

Epiblast stem cells (EpiSCs), which are pluripotent cells isolated from early post-implantation mouse embryos (E5.5), show both similarities and differences compared to mouse embryonic stem cells (mESCs), isolated earlier from the inner cell mass (ICM) of the E3.5 embryo. Previously, we have observed that while chromatin is very dispersed in E3.5 ICM, compact chromatin domains and chromocentres appear in E5.5 epiblasts after embryo implantation. Given that the observed chromatin re-organization in E5.5 epiblasts coincides with an increase in DNA methylation, in this study, we aimed to examine the role of DNA methylation in chromatin re-organization during the in vitro conversion of ESCs to EpiSCs. The requirement for DNA methylation was determined by converting both wild-type and DNA methylation-deficient ESCs to EpiSCs, followed by structural analysis with electron spectroscopic imaging (ESI). We show that the chromatin re-organization which occurs in vivo can be re-capitulated in vitro during the ESC to EpiSC conversion. Indeed, after 7 days in EpiSC media, compact chromatin domains begin to appear throughout the nuclear volume, creating a chromatin organization similar to E5 epiblasts and embryo-derived EpiSCs. Our data demonstrate that DNA methylation is dispensable for this global chromatin re-organization but required for the compaction of pericentromeric chromatin into chromocentres.

Primordial germ cell differentiation of nuclear transfer embryonic stem cells using surface modified electroconductive scaffolds.

A combination of nanotopographical cues and surface modification of collagen and fibronectin is a potential platform in primordial germ cells (PGCs) differentiation. In the present study, the synergistic effect of nanotopography and surface modification on differentiation of nuclear transfer embryonic stem cells (nt-ESCs) toward PGC lineage was investigated. In order to achieve this goal, poly-anyline (PANi) was mix within poly-L-lactic acid (PLLA). Afterward, the random composite mats were fabricated using PLLA and PANi mix solution. The nanofiber topography notably upregulated the expressions of prdm14, mvh and c-kit compared with tissue culture polystyrene (TCP). Moreover, the combination of nanofiber topography and surface modification resulted in more enhancement of PGCs differentiation compared with non-modified nanofibrous scaffold. Additionally, gene expression results showed that mvh and c-kit were expressed at higher intensity in cells exposed to collagen and fibronectin rather than collagen or fibronectin solitary. These results demonstrated the importance of combined effect of collagen and fibronectin in order to develop a functional extracellular matrix (ECM) mimic in directing stem cell fate and the potential of such biofunctional scaffolds for treatment of infertility.