Proteomic profiling of mesenchymal stem cell-derived extracellular vesicles: Impact of isolation methods on protein cargo.

Extracellular vesicles (EVs) are nanosized vesicles with a lipid bilayer that are secreted by cells and play a critical role in cell-to-cell communication. Despite the promising reports regarding their diagnostic and therapeutic potential, the utilization of EVs in the clinical setting is limited due to insufficient information about their cargo and a lack of standardization in isolation and analysis methods. Considering protein cargos in EVs as key contributors to their therapeutic potency, we conducted a tandem mass tag (TMT) quantitative proteomics analysis of three subpopulations of mesenchymal stem cell (MSC)-derived EVs obtained through three different isolation techniques: ultracentrifugation (UC), high-speed centrifugation (HS), and ultracentrifugation on sucrose cushion (SU). Subsequently, we checked EV marker expression, size distribution, and morphological characterization, followed by bioinformatic analysis. The bioinformatic analysis of the proteome results revealed that these subpopulations exhibit distinct molecular and functional characteristics. The choice of isolation method impacts the proteome of isolated EVs by isolating different subpopulations of EVs. Specifically, EVs isolated through the high-speed centrifugation (HS) method exhibited a higher abundance of ribosomal and mitochondrial proteins. Functional apoptosis assays comparing isolated mitochondria with EVs isolated through different methods revealed that HS-EVs, but not other EVs, induced early apoptosis in cancer cells. On the other hand, EVs isolated using the sucrose cushion (SU) and ultracentrifugation (UC) methods demonstrated a higher abundance of proteins primarily involved in the immune response, cell-cell interactions and extracellular matrix interactions. Our analyses unveil notable disparities in proteins and associated biological functions among EV subpopulations, underscoring the importance of meticulously selecting isolation methods and resultant EV subpopulations based on the intended application.

Y Chromosome Genes May Play Roles in the Development of Neural Rosettes from Human Embryonic Stem Cells.

BACKGROUND: The human Y chromosome harbors genes that are mainly involved in the growth, development, sexual dimorphism, and spermatogenesis process. Despite many studies, the function of the male-specific region of the Y chromosome (MSY) awaits further clarification, and a cell-based approach can help in this regard. RESULTS: In this study, we have developed four stable transgenic male embryonic stem cell (ESCs) lines that can overexpress male-specific genes HSFY1, RBMY1A1, RPS4Y1, and SRY. As a proof of principle, we differentiated one of these cell lines (RPS4Y1 over-expressing ESCs) to the neural stem cell (rosette structure) and characterized them based on the expression level of lineage markers. RPS4Y1 expression in the Doxycycline-treated group was significantly higher than control groups at transcript and protein levels. Furthermore, we found Doxycycline-treated group had a higher differentiation efficiency than the untreated control groups. CONCLUSIONS: Our results suggest that the RPS4Y1 gene may play a critical role in neurogenesis. Also, the generated transgenic ESC lines can be widely employed in basic and preclinical studies, such as sexual dimorphism of neural and cardiac functions, the development of cancerous and non-cancerous disease models, and drug screening.

Y chromosome is moving out of sex determination shadow.

Although sex hormones play a key role in sex differences in susceptibility, severity, outcomes, and response to therapy of different diseases, sex chromosomes are also increasingly recognized as an important factor. Studies demonstrated that the Y chromosome is not a 'genetic wasteland' and can be a useful genetic marker for interpreting various male-specific physiological and pathophysiological characteristics. Y chromosome harbors male­specific genes, which either solely or in cooperation with their X-counterpart, and independent or in conjunction with sex hormones have a considerable impact on basic physiology and disease mechanisms in most or all tissues development. Furthermore, loss of Y chromosome and/or aberrant expression of Y chromosome genes cause sex differences in disease mechanisms. With the launch of the human proteome project (HPP), the association of Y chromosome proteins with pathological conditions has been increasingly explored. In this review, the involvement of Y chromosome genes in male-specific diseases such as prostate cancer and the cases that are more prevalent in men, such as cardiovascular disease, neurological disease, and cancers, has been highlighted. Understanding the molecular mechanisms underlying Y chromosome-related diseases can have a significant impact on the prevention, diagnosis, and treatment of diseases.

Inner retinal injury in experimental glaucoma is prevented upon AAV mediated Shp2 silencing in a caveolin dependent manner.

SH2 domain containing tyrosine phosphatase 2 (Shp2; PTPN11) regulates several intracellular pathways downstream of multiple growth factor receptors. Our studies implicate that Shp2 interacts with Caveolin-1 (Cav-1) protein in retinal ganglion cells (RGCs) and negatively regulates BDNF/TrkB signaling. This study aimed to investigate the mechanisms underlying the protective effects of shp2 silencing in the RGCs in glaucomatous conditions. Methods: Shp2 was silenced in the Cav-1 deficient mice and the age matched wildtype littermates using adeno-associated viral (AAV) constructs. Shp2 expression modulation was performed in an acute and a chronic mouse model of experimental glaucoma. AAV2 expressing Shp2 eGFP-shRNA under a strong synthetic CAG promoter was administered intravitreally in the animals' eyes. The contralateral eye received AAV-eGFP-scramble-shRNA as control. Animals with Shp2 downregulation were subjected to either microbead injections or acute ocular hypertension experimental paradigm. Changes in inner retinal function were evaluated by measuring positive scotopic threshold response (pSTR) while structural and biochemical alterations were evaluated through H&E staining, western blotting and immunohistochemical analysis of the retinal tissues. Results: A greater loss of pSTR amplitudes was observed in the WT mice compared to Cav-1-/- retinas in both the models. Silencing of Shp2 phosphatase imparted protection against inner retinal function loss in chronic glaucoma model in WT mice. The functional rescue also translated to structural preservation of ganglion cell layer in the chronic glaucoma condition in WT mice which was not evident in Cav-1-/- mice retinas. Conclusions: This study indicates that protective effects of Shp2 ablation under chronic experimental glaucoma conditions are dependent on Cav-1 in the retina, suggesting in vivo interactions between the two proteins.

The quest of cell surface markers for stem cell therapy.

Stem cells and their derivatives are novel pharmaceutics that have the potential for use as tissue replacement therapies. However, the heterogeneous characteristics of stem cell cultures have hindered their biomedical applications. In theory and practice, when cell type-specific or stage-specific cell surface proteins are targeted by unique antibodies, they become highly efficient in detecting and isolating specific cell populations. There is a growing demand to identify reliable and actionable cell surface markers that facilitate purification of particular cell types at specific developmental stages for use in research and clinical applications. The identification of these markers as very important members of plasma membrane proteins, ion channels, transporters, and signaling molecules has directly benefited from proteomics and tools for proteomics-derived data analyses. Here, we review the methodologies that have played a role in the discovery of cell surface markers and introduce cutting edge single cell proteomics as an advanced tool. We also discuss currently available specific cell surface markers for stem cells and their lineages, with emphasis on the nervous system, heart, pancreas, and liver. The remaining gaps that pertain to the discovery of these markers and how single cell proteomics and identification of surface markers associated with the progenitor stages of certain terminally differentiated cells may pave the way for their use in regenerative medicine are also discussed.

Proteome analysis of endometrial tissue from patients with PCOS reveals proteins predicted to impact the disease.

Polycystic ovary syndrome (PCOS) is a complex disease that causes an ovulatory infertility in approximately 10% of reproductive-age women. We searched for candidate proteins that might contribute to endometrial receptivity defects in PCOS patients, and result in adverse reproductive outcomes. Shotgun proteomics approach was used to investigate the proteome profile of the endometrium at the luteal phase in PCOS patients compared to healthy fertile individuals. Biological process and pathway analyses were conducted to categorize the proteins with differential expressions. Confirmation was performed for a number of proteins via immunoblotting in new samples. 150 proteins with higher abundance, and 46 proteins with lower abundance were identified in the endometrial tissue from PCOS patients compared to healthy fertile individuals. The proteins with higher abundance were enriched in protein degradation, cell cycle, and signaling cascades. Proteins with lower abundance in PCOS patients were enriched in extracellular matrix (ECM) composition and function, as well as the salvage pathway of purine biosynthesis. Metabolism was the most affected biological process with over 100 up-regulated, and approximately 30 down-regulated proteins. Our results indicate significant imbalances in metabolism, proteasome, cell cycle, ECM related proteins, and signaling cascades in endometrial tissue of PCOS, which may contribute to poor reproductive outcomes in these patients. We postulate that the endometria in PCOS patients may not be well-differentiated and synchronized for implantation. Possible roles of the above-mentioned pathways that underlie implantation failure in PCOS will be discussed. Our findings need to be confirmed in larger populations.

SOX2 protein transduction directly converts human fibroblasts into oligodendrocyte-like cells.

Oligodendrocyte precursor cells (OPCs) are ideal therapeutic cells for treatment of spinal cord injuries and diseases that affect myelin. However, it is necessary to generate a cell population with a low risk of teratoma formation and oncogenesis from a patient's somatic cells. In this study, we investigated the direct reprogramming of fibroblasts to oligodendrocyte-like cells in one step with a safe non-genetic delivery method that used protein transduction. Cell morphology and the lineage-specific marker expression profile indicated that human foreskin fibroblasts (HFFs) were converted into oligodendrocyte-like cells by the application of pluripotency factors and the use of a permissible induction medium. Our data demonstrated that SOX2 was sufficient to directly drive OPC fate conversion from HFF by a genetic-free approach. Therefore, this work has provided a strategy to OPC reprogramming by a non-integrating approach for future use in disease modeling and may ultimately provide applications for patient-specific cell-based regenerative medicine.

Discovery of Novel Cell Surface Markers for Purification of Embryonic Dopamine Progenitors for Transplantation in Parkinson's Disease Animal Models.

Despite the progress in safety and efficacy of cell replacement therapy with pluripotent stem cells (PSCs), the presence of residual undifferentiated stem cells or proliferating neural progenitor cells with rostral identity remains a major challenge. Here we report the generation of a LIM homeobox transcription factor 1 alpha (LMX1A) knock-in GFP reporter human embryonic stem cell (hESC) line that marks the early dopaminergic progenitors during neural differentiation to find reliable membrane protein markers for isolation of midbrain dopaminergic neurons. Purified GFP positive cells in vitro exhibited expression of mRNA and proteins that characterized and matched the midbrain dopaminergic identity. Further quantitative proteomics analysis of enriched LMX1A+ cells identified several membrane-associated proteins including a polysialylated embryonic form of neural cell adhesion molecule (PSA-NCAM) and contactin 2 (CNTN2), enabling prospective isolation of LMX1A+ progenitor cells. Transplantation of human-PSC-derived purified CNTN2+ progenitors enhanced dopamine release from transplanted cells in the host brain and alleviated Parkinson's disease-related phenotypes in animal models. This study establishes an efficient approach for purification of large numbers of human-PSC-derived dopaminergic progenitors for therapeutic applications.

Distinct changes in the proteome profile of endometrial tissues in polycystic ovary syndrome compared with healthy fertile women.

RESEARCH QUESTION: What is the molecular basis of infertility related to uterine dysfunction in women with polycystic ovary syndrome (PCOS)? DESIGN: In this study, differences in protein expression between PCOS and normal endometrium were identified using a proteomic approach based on two-dimensional electrophoresis (2-DE) coupled with mass spectrometry (MS). The proteome of endometrium were analysed during the proliferative (on day 2 or 3 before ovulation, n = 6) and luteal phases (on day 3-5 after ovulation, n = 6) from healthy women and PCOS patients (12-14 days after spontaneous bleeding, n = 12). The differentially expressed proteins were categorized based on the biological process using the DAVID bioinformatics resources. RESULTS: Over 803 reproducible protein spots were detected on gels, and 150 protein spots showed different intensities between PCOS and normal women during the proliferative and luteal phases. MS analysis detected 70 proteins out of 150 spots. For four of the 70 proteins, 14-3-3 protein, annexin A5, SERPINA1 and cathepsin D, 2-DE results were validated and localized by Western blot and immunohistochemistry, respectively, and their gene expression profiles were confirmed by real-time quantitative PCR. The obtained results corresponded to the proteomic analysis. The differentially expressed proteins identified are known to be involved in apoptosis, oxidative stress, inflammation and the cytoskeleton. CONCLUSIONS: The processes related to the differentially expressed proteins play important roles in fecundity and fecundability. The present study may reveal the cause of various endometrial aberrations as a limiting factor for achieving pregnancy in PCOS women.

Prospective Isolation of ISL1+ Cardiac Progenitors from Human ESCs for Myocardial Infarction Therapy.

The LIM-homeodomain transcription factor ISL1 marks multipotent cardiac progenitors that give rise to cardiac muscle, endothelium, and smooth muscle cells. ISL1+ progenitors can be derived from human pluripotent stem cells, but the inability to efficiently isolate pure populations has limited their characterization. Using a genetic selection strategy, we were able to highly enrich ISL1+ cells derived from human embryonic stem cells. Comparative quantitative proteomic analysis of enriched ISL1+ cells identified ALCAM (CD166) as a surface marker that enabled the isolation of ISL1+ progenitor cells. ALCAM+/ISL1+ progenitors are multipotent and differentiate into cardiomyocytes, endothelial cells, and smooth muscle cells. Transplantation of ALCAM+ progenitors enhances tissue recovery, restores cardiac function, and improves angiogenesis through activation of AKT-MAPK signaling in a rat model of myocardial infarction, based on cardiac MRI and histology. Our study establishes an efficient method for scalable purification of human ISL1+ cardiac precursor cells for therapeutic applications.

MicroRNA-340 inhibits the proliferation and promotes the apoptosis of colon cancer cells by modulating REV3L.

DNA Directed Polymerase Zeta Catalytic Subunit (REV3L) has recently emerged as an important oncogene. Although the expressions of REV3L are similar in normal and cancer cells, several mutations in REV3L have been shown to play important roles in cancer. These mutations cause proteins misfolding and mislocalization, which in turn alters their interactions and biological functions. miRNAs play important regulatory roles during the progression and metastasis of several human cancers. This study was undertaken to determine how changes in the location and interactions of REV3L regulate colon cancer progression. REV3L protein mislocalization confirmed from the immunostaining results and the known interactions of REV3L was found to be broken as seen from the PLA assay results. The mislocalized REV3L might interact with new proteins partners in the cytoplasm which in turn may play role in regulating colon cancer progression. hsa-miR-340 (miR-340), a microRNA down-regulated in colon cancer, was used to bind to and downregulate REV3L, and found to control the proliferation and induce the apoptosis of colon cancer cells (HCT-116 and DLD-1) via the MAPK pathway. Furthermore, this down-regulation of REV3L also diminished colon cancer cell migration, and down-regulated MMP-2 and MMP-9. Combined treatment of colon cancer cells with miR-340 and 5-FU enhanced the inhibitory effects of 5-FU. In addition, in vivo experiments conducted on nude mice revealed tumor sizes were smaller in a HCT-116-miR-340 injected group than in a HCT-116-pCMV injected group. Our findings suggest mutations in REV3L causes protein mislocalization to the cytoplasm, breaking its interaction and is believed to form new protein interactions in cytoplasm contributing to colon cancer progression. Accordingly, microRNA-340 appears to be a good candidate for colon cancer therapy.

Low Focal Adhesion Signaling Promotes Ground State Pluripotency of Mouse Embryonic Stem Cells.

Mouse embryonic stem cells (mESCs) can be maintained in a pluripotent state when cultured with 2 inhibitors (2i) of extracellular signal-regulated kinase (MEK) and glycogen synthase kinase-3 (GSK3), and Royan 2 inhibitors (R2i) of FGF4 and TGFß. The molecular mechanisms that control ESC self-renewal and pluripotency are more important for translating stem cell technologies to clinical applications. In this study, we used the shotgun proteomics technique to compare the proteome of the ground state condition (R2i- and 2i-grown cells) to that of serum. Out of 1749 proteins identified, 171 proteins were differentially expressed (p < 0.05) in the 2i, R2i, and serum samples. Gene ontology (GO) analysis of differentially abundant proteins showed that the focal adhesion signaling pathway significantly down-regulated under ground state conditions. mESCs had highly adhesive attachment under the serum condition, whereas in the 2i and R2i culture conditions, a loss of adhesion was observed and the cells were rounded and grew in compact colonies on gelatin. Quantitative RT-PCR showed reduced expression of the integrins family in the 2i and R2i conditions. The serum culture had more prominent phosphorylation of focal adhesion kinase (FAK) compared to 2i and R2i cultures. Activity of the extracellular signal-regulated kinase (ERK)1/2 decreased in the 2i and R2i cultures compared to serum. Activation of integrins by Mn2+ in the 2i and R2i cultures resulted in reduced Nanog and increased the expression of lineage marker genes. In this study, we demonstrated that reduced focal adhesion enabled mESCs to be maintained in an undifferentiated and pluripotent state.

Transient Activation of Reprogramming Transcription Factors Using Protein Transduction Facilitates Conversion of Human Fibroblasts Toward Cardiomyocyte-Like Cells.

Derivation of cardiomyocytes directly from patients' own fibroblasts could offer a new therapeutic approach for those with ischemic heart disease. An essential step toward clinical application is to establish safe conversion of human fibroblasts into a cardiac fate. Here we aimed to efficiently and safely generate cardiomyocytes from human fibroblasts by direct delivery of reprogramming recombinant cell permeant form of reprogramming proteins followed by cardio-inductive signals. Human fetal and adult fibroblasts were transiently exposed to transactivator of transcription-fused recombinant OCT4, SOX2, KLF4 and c-MYC for 2 weeks and then were directly differentiated toward protein-induced cardiomyocyte-like cells (p-iCLCs) in a cardiac fate niche, carried out by treatment with a set of cardiogenic small molecules (sequential treatment of Chir, and IWP-2, SB431542 and purmorphamine). The cells showed cardiac phenotype over a period of 3 weeks without first undergoing reprogramming into or through a pluripotent intermediate, shown by lack of expression of key pluripotency markers. p-iCLCs exhibited cardiac features at both the gene and protein levels. Our study provides an alternative method for the generation of p-iCLCs which shortcut reprogramming toward allogeneic cardiomyocytes in a safe and efficient manner and could facilitate generation of genetic material-free cardiomyocytes.

Proteome analysis of human embryonic stem cells organelles.

As the functions of proteins are associated with their cellular localization, the comprehensive sub-cellular proteome knowledge of human embryonic stem cells (hESCs) is indispensable for ensuring a therapeutic effect. Here, we have utilized a sub-cellular proteomics approach to analyze the localization of proteins in the nucleus, mitochondria, crude membrane, cytoplasm, heavy and light microsomes. Out of 2002 reproducibly identified proteins, we detected 762 proteins in a single organelle whereas 160 proteins were found in all sub-cellular fractions. We verified the localization of identified proteins through databases and discussed the consistency of the obtained results. With regards to the ambiguity in the definition of a membrane protein, we tried to clearly define the plasma membrane, peripheral membrane and membrane proteins by annotation of these proteins in databases, along with predictions of transmembrane helices. Among ten enriched signaling pathways highlighted in our results, non-canonical Wnt signaling were analyzed in greater detail. The functions of three novel hESC membrane proteins (ERBB4, GGT1 and ZDHHC13) have been assessed in terms of pluripotency. Our report is the most comprehensive for organellar proteomics of hESCs. SIGNIFICANCE: Mass spectrometric identification of proteins using a TripleTOF 5600 from nucleus, mitochondria, crude membrane, cytoplasm, heavy and light microsomal fractions highlighted the significance of the non-canonical Wnt signaling in human embryonic stem cells.

Red/ox states of human protein disulfide isomerase regulate binding affinity of 17 beta-estradiol.

Human protein disulfide isomerase (hPDI) is a key redox-regulated thiol-containing protein operating as both oxidoreductase and molecular chaperone in the endoplasmic reticulum of cells. hPDI thiol-disulfide interchange reactions lead to the adoption of two distinct red/ox conformations with different substrate preferences. hPDI also displays high binding capacity for some endogenous steroid hormones including 17 beta-estradiol (E2) and thus contributes to the regulation of their intracellular concentration, storage and actions. The primary focus of this study was to investigate the impact of E2 binding on functional activity of recombinant hPDI. Then, we examined the effect of E2 binding on structural alteration of hPDI red/ox conformations and its influence on affinity and position of interaction using experimental and computational analysis. Our results revealed that interaction of one E2 per each hPDI molecule led to the inhibition of hPDI reductase activity and conformational changes in both oxidation states. Mutually, E2-binding position were also redox-regulated with higher affinity in oxidized hPDI compare to the reduced form. The importance of histidine-256 protonation states in distinct binding preferences of E2 were also demonstrated in hPDI red/ox conformations. These findings might pave the way for better understanding of the mechanisms behind the redox-dependent hormone-binding activity of hPDI.

Advanced glycation end-products produced systemically and by macrophages: A common contributor to inflammation and degenerative diseases.

Advanced glycation end products (AGEs) and their receptor have been implicated in the progressions of many intractable diseases, such as diabetes and atherosclerosis, and are also critical for pathologic changes in chronic degenerative diseases, such as Alzheimer's disease, Parkinson's disease, and alcoholic brain damage. Recently activated macrophages were found to be a source of AGEs, and the most abundant form of AGEs, AGE-albumin excreted by macrophages has been implicated in these diseases and to act through common pathways. AGEs inhibition has been shown to prevent the pathogenesis of AGEs-related diseases in human, and therapeutic advances have resulted in several agents that prevent their adverse effects. Recently, anti-inflammatory molecules that inhibit AGEs have been shown to be good candidates for ameliorating diabetic complications as well as degenerative diseases. This review was undertaken to present, discuss, and clarify current understanding regarding AGEs formation in association with macrophages, different diseases, therapeutic and diagnostic strategy and links with RAGE inhibition.

Two Splice Variants of Y Chromosome-Located Lysine-Specific Demethylase 5D Have Distinct Function in Prostate Cancer Cell Line (DU-145).

One of the major objectives of the Human Y Chromosome Proteome Project is to characterize sets of proteins encoded from the human Y chromosome. Lysine (K)-specific demethylase 5D (KDM5D) is located on the AZFb region of the Y chromosome and encodes a JmjC-domain-containing protein. KDM5D, the least well-documented member of the KDM5 family, is capable of demethylating di- and trimethyl H3K4. In this study, we detected two novel splice variants of KDM5D with lengths of 2650bp and 2400bp that correspond to the 100 and 80 kDa proteins in the human prostate cancer cell line, DU-145. The knockdown of two variants using the short interfering RNA (siRNA) approach increased the growth rate of prostate cancer cells and reduced cell apoptosis. To explore the proteome pattern of the cells after KDM5D downregulation, we applied a shotgun label-free quantitative proteomics approach. Of 820 proteins present in all four replicates of two treatments, the abundance of 209 proteins changed significantly in response to KDM5D suppression. Of these, there were 102 proteins observed to be less abundant and 107 more abundant in KDM5D knockdown cells compared with control cells. The results revealed that KDM5D knockdown altered the abundance of proteins involved in RNA processing, protein synthesis, apoptosis, the cell cycle, and growth and proliferation. In conjunction, these results provided new insights into the function of KDM5D and its splice variants. The proteomics data are available at PRIDE with ProteomeXchange identifier PXD000416.

DDX3Y, a Male-Specific Region of Y Chromosome Gene, May Modulate Neuronal Differentiation.

Although it is apparent that chromosome complement mediates sexually dimorphic expression patterns of some proteins that lead to functional differences, there has been insufficient evidence following the manipulation of the male-specific region of the Y chromosome (MSY) gene expression during neural development. In this study, we profiled the expression of 23 MSY genes and 15 of their X-linked homologues during neural cell differentiation of NTERA-2 human embryonal carcinoma cell line (NT2) cells in three different developmental stages using qRT-PCR, Western blotting, and immunofluorescence. The expression level of 12 Y-linked genes significantly increased over neural differentiation, including RBMY1, EIF1AY, DDX3Y, HSFY1, BPY2, PCDH11Y, UTY, RPS4Y1, USP9Y, SRY, PRY, and ZFY. We showed that siRNA-mediated knockdown of DDX3Y, a DEAD box RNA helicase enzyme, in neural progenitor cells impaired cell cycle progression and increased apoptosis, consequently interrupting differentiation. Label-free quantitative shotgun proteomics based on a spectral counting approach was then used to characterize the proteomic profile of the cells after DDX3Y knockdown. Among 917 reproducibly identified proteins detected, 71 proteins were differentially expressed following DDX3Y siRNA treatment compared with mock treated cells. Functional grouping indicated that these proteins were involved in cell cycle, RNA splicing, and apoptosis, among other biological functions. Our results suggest that MSY genes may play an important role in neural differentiation and demonstrate that DDX3Y could play a multifunctional role in neural cell development, probably in a sexually dimorphic manner.

Isoform-Level Gene Expression Profiles of Human Y Chromosome Azoospermia Factor Genes and Their X Chromosome Paralogs in the Testicular Tissue of Non-Obstructive Azoospermia Patients.

The human Y chromosome has an inevitable role in male fertility because it contains many genes critical for spermatogenesis and the development of the male gonads. Any genetic variation or epigenetic modification affecting the expression pattern of Y chromosome genes may thus lead to male infertility. In this study, we performed isoform-level gene expression profiling of Y chromosome genes within the azoospermia factor (AZF) regions, their X chromosome counterparts, and few autosomal paralogues in testicular biopsies of 12 men with preserved spermatogenesis and 68 men with nonobstructive azoospermia (NOA) (40 Sertoli-cell-only syndrome (SCOS) and 28 premiotic maturation arrest (MA)). This was undertaken using quantitative real-time PCR (qPCR) at the transcript level and Western blotting (WB) and immunohistochemistry (IHC) at the protein level. We profiled the expression of 41 alternative transcripts encoded by 14 AZFa, AZFb, and AZFc region genes (USP9Y, DDX3Y, XKRY, HSFY1, CYORF15A, CYORF15B, KDM5D, EIF1AY, RPS4Y2, RBMY1A1, PRY, BPY2, DAZ1, and CDY1) as well as their X chromosome homologue transcripts and a few autosomal homologues. Of the 41 transcripts, 18 were significantly down-regulated in men with NOA when compared with those of men with complete spermatogenesis. In contrast, the expression of five transcripts increased significantly in NOA patients. Furthermore, to confirm the qPCR results at the protein level, we performed immunoblotting and IHC experiments (based on 24 commercial and homemade antibodies) that detected 10 AZF-encoded proteins. In addition, their localization in testis cell types and organelles was determined. Interestingly, the two missing proteins, XKRY and CYORF15A, were detected for the first time. Finally, we focused on the expression patterns of the significantly altered genes in 12 MA patients with successful sperm retrieval compared to those of 12 MA patients with failed sperm retrieval to predict the success of sperm retrieval in azoospermic men. We showed that HSFY1-1, HSFY1-3, BPY2-1, KDM5C2, RBMX2, and DAZL1 transcripts could be used as potential molecular markers to predict the presence of spermatozoa in MA patients. In this study, we have identified isoform level signature that can be used to discriminate effectively between MA, SCOS, and normal testicular tissues and suggests the possibility of diagnosing the presence of mature sperm cell in azoospermic men to prevent additional testicular sperm extraction (TESE) surgery.

Cellular and molecular characterization of human cardiac stem cells reveals key features essential for their function and safety.

Cell therapy of heart diseases is emerging as one of the most promising known treatments in recent years. Transplantation of cardiac stem cells (CSCs) may be one of the best strategies to cure adult or pediatric heart diseases. As these patient-derived stem cells need to be isolated from small heart biopsies, it is important to select the best isolation method and CSC subpopulation with the best cardiogenic functionality. We employed three different protocols including c-KIT(+) cell sorting, clonogenic expansion, and explants culture to isolate c-KIT(+) cells, clonogenic expansion-derived cells (CEDCs), and cardiosphere-derived cells (CDCs), respectively. Evaluation of isolated CSC characteristics in vitro and after rat myocardial infarction (MI) model transplantation revealed that although c-KIT(+) and CDCs had higher MI regenerative potential, CEDCs had more commitment into cardiomyocytes and needed lower passages that were essential to reach a definite cell count. Furthermore, genome-wide expression analysis showed that subsequent passages caused changes in characteristics of cells, downregulation of cell cycle-related genes, and upregulation of differentiation and carcinogenic genes, which might lead to senescence, commitment, and possible tumorigenicity of the cells. Because of different properties of CSC subpopulations, we suggest that appropriate CSCs subpopulation should be chosen based on their experimental or clinical use.