Hyaluronan Receptor LYVE-1-Expressing Macrophages Maintain Arterial Tone through Hyaluronan-Mediated Regulation of Smooth Muscle Cell Collagen.

The maintenance of appropriate arterial tone is critically important for normal physiological arterial function. However, the cellular and molecular mechanisms remain poorly defined. Here, we have shown that in the mouse aorta, resident macrophages prevented arterial stiffness and collagen deposition in the steady state. Using phenotyping, transcriptional profiling, and targeted deletion of Csf1r, we have demonstrated that these macrophages-which are a feature of blood vessels invested with smooth muscle cells (SMCs) in both mouse and human tissues-expressed the hyaluronan (HA) receptor LYVE-l. Furthermore, we have shown they possessed the unique ability to modulate collagen expression in SMCs by matrix metalloproteinase MMP-9-dependent proteolysis through engagement of LYVE-1 with the HA pericellular matrix of SMCs. Our study has unveiled a hitherto unknown homeostatic contribution of arterial LYVE-1+ macrophages through the control of collagen production by SMCs and has identified a function of LYVE-1 in leukocytes.

Histological, immunohistochemical, and genomic evaluation of excisional and diabetic wounds treated with human Wharton's jelly stem cells with and without a nanocarrier.

We showed in previous studies that human umbilical cord Wharton's jelly stem cells (hWJSCs) improved the healing rates of excisional and diabetic wounds in the mouse model. As an extension of those studies, we report here the more detailed quantitative histological, immunohistochemical, and genomic evaluation of biopsies from those excisional and diabetic wounds in an attempt to understand the mechanisms of the enhanced wound healing aided by hWJSCs. Bright-field microscopic observations and ImageJ software analysis on histological sections of the excisional and diabetic wound biopsies collected at different time points showed that the thickness of the epidermis and dermis, and positive picrosirius-red stained areas for collagen, were significantly greater in the presence of hWJSCs compared with controls (P < 0.05). Immunohistochemistry of the diabetic wound biopsies showed increased positive staining for the vascular endothelial marker CD31 and cell proliferation marker Ki67 in the presence of hWJSCs and its conditioned medium (hWJSC-CM). Quantitative real-time polymerase chain reaction showed upregulation of groups of genes involved in extracellular matrix regulation, collagen biosynthesis, angiogenesis, antifibrosis, granulation, and immunomodulation in the presence of hWJSCs. Taken together, the results demonstrated that hWJSCs and hWJSC-CM that contains the paracrine secretions of hWJSCs, enhance the healing of excisional and diabetic wounds via re-epithelialization, collagen deposition, angiogenesis, and immunomodulation. The inclusion of an Aloe vera-polycaprolactone (AV/PCL) nanocarrier did not significantly change the effect of the hWJSCs. However, the topical application of an AV/PCL nanocarrier impregnated with hWJSCs is convenient and less invasive than the administration of hWJSC injections into wounds.

Manufacturing of human Wharton's jelly stem cells for clinical use: selection of serum is important.

BACKGROUND: Human Wharton's jelly-derived mesenchymal stromal cells (hWJSCs) have gained considerable attention for their use in cell therapy. Many of these applications would require manufacturing of millions of hWJSCs. It is, therefore, necessary to develop a Good Manufacturing Practice (GMP)-compliant hWJSC expansion protocol, allowing the generation of a large quantity of cells to meet both clinical and regulatory requirements. Here, we compared human platelet lysate (HPL) and human serum (HS) in supporting clinical-grade hWJSC expansion. METHODS: hWJSCs were successfully isolated from six different umbilical cords using GMP-compliant dissociation enzymes. Freshly isolated hWJSCs were cultured in media supplemented with 10% of one of the following sera: fetal bovine serum (FBS), HPL and HS. Properties of the expanded hWJSCs were analyzed. RESULTS: We showed that GMP-compliant dissociation enzymes were as efficient as research-grade dissociation enzymes in isolating hWJSCs. hWJSC fresh cell yield and cell viability using HPL and HS supplementations were at greater advantages than FBS. Moreover, hWJSCs expanded in HPL and HS supplementations not only preserved classical MSCs phenotypes and differentiation potential to adipocytes, osteocytes and chondrocytes, they also enhanced the migration of skin fibroblasts. However, HS, unlike HPL, did not alter immunogenicity properties of hWJSCs. hWJSCs expanded in HS supplementation also exerted greater immunosuppressive action in inhibiting T-cell proliferation and increased extracellular matrix (ECM) gene expression, making them useful in tissue repair clinical application. CONCLUSION: Our findings indicate that HS can be considered as a promising and safer alternative to FBS, and should be recommended for clinical-grade expansion of hWJSCs.

Biology of human primitive erythroblasts for application in noninvasive prenatal diagnosis.

OBJECTIVE: Human primitive erythroblasts produced during early embryogenesis have been found in maternal circulation at early gestation and are considered good target cells for noninvasive prenatal diagnosis. We aimed to gain a better understanding of the biology of primitive erythroblasts and maximize their potential utility for noninvasive prenatal diagnosis. METHODS: Cells were obtained from first trimester human placental tissues. Biological properties including surface antigen composition, differentiation, proliferation, enucleation, and degeneration were studied as gestation progressed. A microdroplet culture system was developed to observe the behavior of these cells in vitro. RESULTS: Histology showed that primitive erythroblasts undergo maturation from polychromatic to orthochromatic erythroblasts and can differentiate spontaneously in vitro. Cell surface markers and nuclear gene expression suggest that the cells do not possess stemness properties, despite being primitive in nature. They have limited proliferative activity and highly deacetylated chromatin, but a microdroplet culture system can prolong their viability under normoxic conditions. No apoptosis was seen by 11 weeks' gestation, and there was no enucleation in vitro. CONCLUSION: These properties confirm that viable cells with intact nuclei can be obtained at very early gestation for genetic analysis.

Tissues Derived From Reprogrammed Wharton's Jelly Stem Cells of the Umbilical Cord Provide an Ideal Platform to Study the Effects of Glucose, Zika Virus, and Other Agents on the Fetus.

The infants of mothers with gestational diabetes mellitus (GDM) have an increased risk of metabolic and cardiovascular disease. It has been difficult to study the direct effects of maternal hyperglycemia on the fetus because of inaccessibility of fetal tissues. The development of tissues that simulate the function of fetal organs using stem cell technology provides an unprecedented opportunity to study this disorder. Stem cells in the Wharton's jelly of the umbilical cord (hWJSCs), possess unique properties that are different from other stem cells. They are primitive, present in large numbers, non-tumorigenic, hypoimmunogenic, tumoricidal, and carry a genetic signature that represents the fetus. They are multipotent but their differentiation into functional pancreatic and cardiovascular tissues has been challenging. We have been able to reprogram hWJSCs from normal and GDM cords into induced pluripotent stem cells (iPSCs) from which a variety of functional fetal tissues including insulin-producing and cardiovascular tissues could be derived. Such tissues from reprogrammed hWJSCs of normal and GDM cords that physiologically and genetically mimic the fetus of the diabetic or non-diabetic mother are an ideal platform to study the effects of glucose, the Zika virus, and other harmful agents on the fetus. The immature stemness phenotype of hWJSCs, easy accessibility, availability in large numbers without the need for propagation, and lower risk of accumulation of epigenetic mutations make them the most attractive candidate over other umbilical cord cell types for reprogramming. Additionally, some of their beneficial genes may be retained in memory in the iPSCs derived from them. J. Cell. Biochem. 118: 437-441, 2017. © 2016 Wiley Periodicals, Inc.

Freezing of Fresh Wharton's Jelly From Human Umbilical Cords Yields High Post-Thaw Mesenchymal Stem Cell Numbers for Cell-Based Therapies.

Some cord blood banks freeze entire pieces of UC (mixed cord, MC) which after post-thaw yields mixed heterogeneous populations of mesenchymal stem cells (MSCs) from all its microanatomical compartments. Freezing of such entire tissues results in sub-optimal post-thaw cell recovery because of poor cryoprotectant diffusion and intracellular ice-formation, heat and water transport issues, and damage to intercellular junctions. To develop a simple method of harvesting pure homogeneous MSCs for cord blood banks, we compared the post-thaw behavior of three groups of frozen UC tissues: (i) freshly harvested WJ without cell separation; (ii) MSCs isolated from WJ (WJSC); and (iii) MC, WJ, and WJSC produced high post-thaw cell survival rates (93.52 ± 6.12% to 90.83 ± 4.51%) and epithelioid monolayers within 24 h in primary culture whereas post-thaw MC explants showed slow growth with mixed epithelioid and fibroblastic cell outgrowths after several days. Viability and proliferation rates of post-thawed WJ and hWJSC were significantly greater than MC. Post-thaw WJ and WJSC produced significantly greater CD24(+) and CD108(+) fluorescence intensities and significantly lower CD40(+) contaminants. Post-thaw WJ and WJSC produced significantly lesser annexin-V-positive and sub-G1 cells and greater degrees of osteogenic and chondrogenic differentiation compared to MC. qRT-PCR analysis of post-thaw MC showed significant decreases in anti-apoptotic gene expression (SURVIVIN, BCL2) and increases in pro-apoptotic (BAX) and cell cycle regulator genes (P53, P21, ROCK 1) compared to WJ and WJSC. We conclude that freezing of fresh WJ is a simple and reliable method of generating large numbers of clinically utilizable MSCs for cell-based therapies.

Human Umbilical Cord Wharton's Jelly Stem Cell Conditioned Medium Induces Tumoricidal Effects on Lymphoma Cells Through Hydrogen Peroxide Mediation.

Several groups have reported that human umbilical cord Wharton's jelly stem cells (hWJSCs) possess unique tumoricidal properties against many cancers. However, the exact mechanisms as to how hWJSCs inhibit tumor growth are not known. Recent evidence suggests that exposure of cancer cells to high hydrogen peroxide (H2 O2 ) levels from H2 O2 -releasing drugs causes their death. We therefore explored whether the tumoricidal effect of hWJSCs on lymphoma cells was mediated via H2 O2 . We first exposed lymphoma cells to six different molecular weight cut-off (MWCO) concentrates of hWJSC-conditioned medium (hWJSC-CM) (3, 5, 10, 30, 50, 100 kDa) for 48 h. Since, the 3 kDa-MWCO concentrate showed the greatest cell inhibition we then investigated whether the tumoricidal effect of the specific 3 kDa-MWCO concentrate on two different lymphoma cell lines (Ramos and Toledo) was mediated via accumulation of H2 O2 . We used a battery of assays (MTT, propidium iodide, mitochondria membrane potential, apoptosis, cell cycle, oxidative stress enzymes, hydrogen peroxide, mitochondrial superoxide, hydroxyl radical, peroxynitrile anion, and lipid peroxidation) to test this mechanism. The hWJSC-CM-3 kDa MWCO concentrate significantly decreased cell viability and mitochondrial membrane potential and increased cell death and apoptosis in both lymphoma cell lines. There were significant increases in superoxide dismutase with concomitant decreases in glutathione peroxidase, catalase, and thioredoxin peroxidase activities. H2 O2 levels, mitochondrial superoxide, hydroxyl radical, peroxynitrile anion, and lipid peroxidation were also significantly increased in both lymphoma cell lines. The results suggested that the hWJSC-CM-3 kDa MWCO concentrate regulates cellular H2 O2 leading to a tumoricidal effect and may thus be a promising anti-lymphoma agent. J. Cell. Biochem. 117: 2045-2055, 2016. © 2016 Wiley Periodicals, Inc.

Human keloid cell characterization and inhibition of growth with human Wharton's jelly stem cell extracts.

Keloids are firm rubbery growths that grow beyond the boundaries of human wounds and their treatment has met with limited success. Their properties and growth behavior have not been properly characterized and it has been suggested that a benign neoplastic stem cell-like phenotype in an altered cytokine microenvironment drives their uncontrolled cell proliferation. Modification of the stem cell niche may be an attractive approach to its prevention. We studied the growth behavior, stemness, and tumorigenic characteristics of keloid cells in prolonged culture. Since human Wharton's jelly stem cells (hWJSCs) secrete high levels of cytokines and have anti-tumorigenic properties we explored its role on the inhibition of keloid growth in vitro. Keloid cells grew readily in both adherent and sphere culture and expressed high levels of mesenchymal CD and tumor-associated fibroblast (TAF) markers up to passage 10. When they were exposed to repeat doses of hWJSC conditioned medium (hWJSC-CM) and lysate (hWJSC-CL) every 72 h up to 9 days their growth was inhibited with a reduction in CD and TAF marker expression. On Days 3, 6, and 9 treated keloid cells showed linear decreases in cell proliferation (BrdU), increases in Annexin V-FITC and TUNEL-positive cells, interruptions of the cell cycle and inhibition of migration in scratch-wound assays. Immunocytochemistry and qRT-PCR confirmed a significant downregulation of TAF and anti-apoptotic-related gene (SURVIVIN) expression and upregulation of autophagy-related (BAX, ATG5, ATG7, BECLIN-1) gene expression. The results suggest that hWJSCs or molecules secreted by them may be of therapeutic value in the treatment of keloids.

A nanoscaffold impregnated with human wharton's jelly stem cells or its secretions improves healing of wounds.

Wound healing is a major problem in diabetic patients and current methods of treatment have met with limited success. Since skin cell renewal is under the control of mesenchymal stem cells (MSCs) treatment of wounds has been attempted with the application of exogenous bone marrow MSCs (hBMMSCs). However, hBMMSCs have the limitations of painful harvest, low cell numbers and short-lived stemness properties unlike MSCs from the Wharton's jelly of human umbilical cords (hWJSCs). Since nanoscaffolds provide three dimensional architectural patterns that mimic in vivo stem cell niches and aloe vera has antibacterial properties we evaluated the use of an aloe vera-polycaprolactone (AV/PCL) nanoscaffold impregnated with green fluorescent protein (GFP)-labeled hWJSCs (GFP-hWJSCs + AV/PCL) or its conditioned medium (hWJSC-CM + AV/PCL) for healing of excisional and diabetic wounds. In skin fibroblast scratch-wound assays exposed to GFP-hWJSCs + AV/PCL or hWJSC-CM + AV/PCL, fibroblasts migrated significantly faster from edges of scratches into vacant areas together with increased secretion of collagen I and III, elastin, fibronectin, superoxide dismutase, and metalloproteinase-1 (MMP-1) compared to controls. After one application of GFP-hWJSCs + AV/PCL or hWJSC-CM + AV/PCL excisional and diabetic wounds in mice showed rapid wound closure, reepithelialization, and increased numbers of sebaceous glands and hair follicles compared to controls. The same wounds exposed to GFP-hWJSCs + AV/PCL or hWJSC-CM + AV/PCL also showed positive keratinocyte markers (cytokeratin, involucrin, filaggrin) and increased expression of ICAM-1, TIMP-1, and VEGF-A compared to controls. AV/PCL nanoscaffolds in combination with hWJSCs appear to have synergistic benefits for wound healing.

Human Wharton's jelly stem cells and its conditioned medium enhance healing of excisional and diabetic wounds.

Wound healing is a major problem in diabetic patients and current treatments have met with limited success. We evaluated the treatment of excisional and diabetic wounds using a stem cell isolated from the human umbilical cord Wharton's jelly (hWJSC) that shares unique properties with embryonic and adult mesenchymal stem cells. hWJSCs are non-controversial, available in abundance, hypo-immunogenic, non-tumorigenic, differentiate into keratinocytes, and secrete important molecules for tissue repair. When human skin fibroblasts (CCD) in conventional scratch-wound assays were exposed to hWJSC-conditioned medium (hWJSC-CM) the fibroblasts at the wound edges migrated and completely covered the spaces by day 2 compared to controls. The number of invaded cells, cell viability, total collagen, elastin, and fibronectin levels were significantly greater in the hWJSC-CM treatment arm compared to controls (P < 0.05). When a single application of green fluorescent protein (GFP)-labeled hWJSCs (GFP-hWJSCs) or hWJSC-CM was administered to full-thickness murine excisional and diabetic wounds, healing rates were significantly greater compared to controls (P < 0.05). Wound biopsies collected at various time points showed the presence of green GFP-labeled hWJSCs, positive human keratinocyte markers (cytokeratin, involucrin, filaggrin) and expression of ICAM-1, TIMP-1, and VEGF-A. On histology, the GFP-hWJSCs and hWJSC-CM treated wounds showed reepithelialization, increased vascularity and cellular density and increased sebaceous gland and hair follicle numbers compared to controls. hWJSCs showed increased expression of several miRNAs associated with wound healing compared to CCDs. Our studies demonstrated that hWJSCs enhance healing of excisional and diabetic wounds via differentiation into keratinocytes and release of important molecules.

Human Wharton's jelly stem cell conditioned medium and cell-free lysate inhibit human osteosarcoma and mammary carcinoma cell growth in vitro and in xenograft mice.

Human Wharton's jelly stem cells (hWJSCs) were shown to inhibit the growth of human mammary carcinomas. It is not known whether cell-free secretions or lysates of hWJSCs do the same on different cancers. They may be less controversial than cells to regulatory bodies for clinical application. We examined the influence of hWJSC conditioned medium (hWJSC-CM) and cell-free lysate (hWJSC-CL) on two osteosarcoma cell lines (MG-63, SKES-1) in vitro and on human mammary carcinomas in immunodeficient mice. When exposed to hWJSC-CL, increased vacuolations in MG-63 and increased membrane fragmentation in SKES-1 cells were observed, with greater cell death in SKES-1. Exposure of SKES-1 and MG-63 cells to hWJSC-CL showed significant decreases in cell proliferation of 46.48 ± 6.66% and 24.32 ± 5.67% respectively compared to controls. MG-63 and SKES-1 cells were annexin V-FITC positive and SKES-1 TUNEL positive following treatment with hWJSC-CM and hWJSC-CL. MG-63 cells were positive and SKES-1 cells negative for anti-BECLIN-1 and anti-LC3B following treatment with hWJSC-CM and hWJSC-CL. RT-PCR showed that the pro-apoptotic BAX gene and the autophagy-related ATG-5 and BECLIN-1 genes were up-regulated while the anti-apoptotic BCL2 and SURVIVIN genes were down-regulated in MG-63 and SKES-1 cells treated with hWJSC-CM and hWJSC-CL. Injections of hWJSCs and hWJSC-CM into mammary carcinomas in immunodeficient mice resulted in decreased tumor sizes and weights of 24.86 ± 6.05% to 37.03 ± 5.91% and 47.14 ± 7.36% to 55.09 ± 5.87% respectively at 6 weeks compared to controls. hWJSC-CM and hWJSC-CL inhibit mammary carcinoma and osteosarcoma cells via apoptosis and autophagy.

Human umbilical cord Wharton's jelly stem cell (hWJSC) extracts inhibit cancer cell growth in vitro.

Umbilical cord mesenchymal stem cells (MSCs) have been shown to inhibit breast cancer cell growth but it is not known whether this effect is specific to only breast cancer cells. We compared the effects of human Wharton's jelly stem cell (hWJSC) extracts [conditioned medium (hWJSC-CM) and cell lysate (hWJSC-CL)] on breast adenocarcinoma (MDA-MB-231), ovarian carcinoma (TOV-112D), and osteosarcoma (MG-63) cells. The cells were treated with either hWJSC-CM (50%) or hWJSC-CL (15 µg/ml) for 48-72 h and changes in cell morphology, proliferation, cycle, gene expression, migration, and cell death studied. All three cancer cell lines showed cell shrinkage, blebbing, and vacuolations with hWJSC-CL and hWJSC-CM compared to controls. MTT and BrdU assays showed inhibition of cell growth by 2-6% and 30-60%, while Transwell migration assay showed inhibition by 20-26% and 31-46% for hWJSC-CM and hWJSC-CL, respectively, for all three cancer cell lines. Cell cycle assays showed increases in sub-G1 and G2/M phases for all three cancer cell lines suggestive of apoptosis and metaphase arrest. AnnexinV-FITC and TUNEL positive cells seen in TOV-112D and MDA-MB-231 suggested that inhibition was via apoptosis while the presence of anti-BECLIN1 and anti-LC3B antibodies seen with MG-63 indicated autophagy. Upregulation of pro-apoptotic BAX and downregulation of anti-apoptotic BCL2 and SURVIVIN genes were observed in all three cancer cell lines and additionally the autophagy genes (ATG5, ATG7, and BECLIN1) were upregulated in MG-63 cells. hWJSCs possess tumor inhibitory properties that are not specific to breast cancer cells alone and these effects are mediated via agents in its extracts.

Human umbilical cord Wharton's jelly mesenchymal stem cells do not transform to tumor-associated fibroblasts in the presence of breast and ovarian cancer cells unlike bone marrow mesenchymal stem cells.

Human bone marrow mesenchymal stem cells (hBMMSCs) were shown to transform into tumor-associated fibroblasts (TAFs) when in the vicinity of breast cancer tumors and played an important role in tumor enhancement and metastasis. In early human development MSCs migrating from the yolk sac and aorta-gonad-mesonephros (AGM) via the umbilical cord to the placenta and back to the fetal bone marrow were shown to get trapped in the gelatinous Wharton's jelly of the umbilical cord. The common origin of the Wharton's jelly MSCs and the finally homed hBMMSCs prompted us to evaluate whether hWJSCs are also involved in TAF transformation. hWJSCs and hBMMSCs were grown in the presence of breast and ovarian cancer cell conditioned medium (MDA-TCM, TOV-TCM) for 30 days. No changes were observed in the hWJSCs but the hBMMSCs transformed from short to thin long fibroblasts, their proliferation rates increased and CD marker expression decreased. The transformed hBMMSCs showed positive staining for the tumor-associated markers FSP, VEGF, EGF, and Tn-C. Real-time PCR and multiplex luminex bead analysis showed upregulation of TAF-related genes (FSP, FAP, Tn-C, Tsp-1, EGF, bFGF, IL-6, α-SMA, VEGF, and TGF-ß) for hBMMSCs with low expression for hWJSCs. The luciferase assay showed that hWJSCs previously exposed to MDA-TCM or TOV-TCM had no stimulatory growth effect on luciferase-tagged MDA or TOV cells unlike hBMMSCs. The results confirmed that hWJSCs do not transform to the TAF phenotype and may therefore not be associated with enhanced growth of solid tumors making them a safe MSC for cell based therapies.

Extra-embryonic human Wharton's jelly stem cells do not induce tumorigenesis, unlike human embryonic stem cells.

Tumorigenesis is the major obstacle of tissues derived from human embryonic stem cells (ESC) and human induced pluripotent stem cell (IPSC) for transplantation therapy. This prompted a search for other sources of ESC. This study isolated and characterized stem cells from the extra-embryonic human umbilical cord Wharton's jelly (WJSC). These cells are non-controversial, available in abundance, proliferative, multipotent and hypoimmunogenic. However, their tumorigenic potential has not been properly addressed. Their tumour-producing capabilities were compared with human ESC using the immunodeficient mouse model. Unlabelled human ESC+matrigel (2×10(6)cells/site), labelled human WJSC (red fluorescent protein; 5×10(6)cells/site) and unlabelled human WJSC+matrigel (5×10(6)cells/site) were injected via three routes (s.c., i.m. and i.p.). Animals that received human ESC+matrigel developed teratomas in 6 weeks (s.c. 85%; i.m. 75%; i.p. 100%) that contained tissues of ectoderm, mesoderm and endoderm. No animal that received human WJSC developed tumours or inflammatory reactions at the injection sites when maintained for a prolonged period (20 weeks). Human WJSC produced increases in anti-inflammatory cytokines in contrast to human ESC, which increased pro-inflammatory cytokines. Human WJSC, being hypoimmunogenic and non-tumorigenic, have the potential for safe cell-based therapies.

Novel approaches to manipulating foetal cells in the maternal circulation for non-invasive prenatal diagnosis of the unborn child.

Due to the risks to the foetus with invasive prenatal diagnosis, non-invasive prenatal diagnosis (NIPD) is gaining tremendous interest but no reliable method that can be widely used has been developed to date. Manipulation of foetal cells and foetal cell-free genetic material in the maternal blood are two promising approaches being researched. The manipulation of foetal cells in the maternal circulation is more popular as it can provide complete genetic information of the foetus particularly the diagnosis of aneuploidies. However, the foetal cell numbers in the maternal circulation are small and their enrichment and ex vivo culture remain two major challenges for NIPD. Primitive foetal erythroblasts (pFEs) have been considered as a good potential candidate for early first trimester NIPD but their nature, properties and manipulation to provide adequate cell numbers remain a challenging task and several approaches need to be meticulously evaluated. In this review we describe the current status of NIPD and suggest some novel approaches in manipulating pFEs for future clinical application of NIPD. These novel approaches include (1) understanding the pFE enucleation process, (2) enriching pFE numbers by individual pick-up of pFEs from maternal blood using micromanipulation and microdroplet culture, (3) expansion of pFEs using mitogens and (4) decondensation of the pFE nucleus with histone deacetylase (HDAC) inhibitors followed by reprogramming using gene delivery protocols with/without small reprogramming molecules to improve reprogrammed pFE proliferation rates for successful NIPD.

Association of decreased sperm motility and increased seminal plasma IGF-I, IGF-II, IGFBP-2, and PSA levels in infertile men.

PURPOSE: Previous studies have suggested the involvement of serum insulin-like growth factors (IGFs) and IGF binding proteins (IGFBPs) in the regulation of the female reproductive system. Little is known of these peptides in the seminal plasma (SP) of men and their potential effects on fertility. We assessed SP levels of these peptides in infertile men with low sperm motility (asthenozoospermic; AZ) and low sperm counts (oligozoospermic; OZ), its effects on in vivo sperm motility, and whether there is a correlation with aging. METHODS: Twenty-eight infertile men (AZ; n = 18 and OZ; n = 10) and 20 fertile normozoospermic (NZ) men were studied. Seminal plasma IGF-I, IGF-II, IGFBP-2, IGFBP-3, and prostate-specific antigen (PSA) levels were measured, and spermatozoa mRNA transcript patterns were examined. RESULTS: Asthenozoospermic men had higher SP IGF-I, IGF-II, IGFBP-2, and PSA levels than NZ and OZ men, whereas SP IGFBP-3 levels were similar between the three groups. Sperm count positively correlated with SP IGF-I, IGF-II, and IGFBP-2; sperm motility negatively correlated with SP IGF-II and IGFBP-2; and age correlated positively with SP IGF-II. The expression of IGF-I and IGF-II mRNA and mRNA receptors was detectable, but no variations in transcript levels were noted. CONCLUSION: Decreased sperm motility, but not sperm count, in infertile AZ men is associated with increased SP IGF-I, IGF-II, IGFBP-2, and PSA levels. Changes in SP IGFs and their interactions with IGFBPs and IGF receptors, and PSA levels suggest a role of these SP peptides in modulating sperm motility and possibly prostate disease development in aging men.

Teratomas from pluripotent stem cells: A clinical hurdle.

Although basic research on human embryonic stem cells (hESCs) at the laboratory bench has progressed with enviable speed there has been little head way in terms of its clinical application. A look at the Internet however shows several stem cell clinics worldwide offering direct transplantation of undifferentiated hESCs to patients for the cure of a variety of diseases before bona fide evidence-based results can be demonstrated from large controlled studies. This raises concern because reliable protocols have to be first developed to resolve the three major hurdles delaying clinical trials such as inadequate cell numbers, immunorejection and tumorigenesis. Cell expansion methods using bioreactors, rotary culture and mitotic agents have now been developed to generate stem cell derivatives in large numbers. The problem of immunorejection can now be overcome with the development of indirect and direct reprogramming protocols to personalize tissues to patients (human induced pluripotent stem cells, hiPSCs; nuclear transfer stem cells, NTSCs; induced neuronal cells, iN). However, hESC, hiPSC, and NTSCs being pluripotent have the disadvantage of teratoma formation in vivo which has to be carefully addressed so as to provide safe stem cell based therapies to the patient. This review addresses the issue of tumorigenesis and discusses approaches by which this concern may be overcome and at the same time emphasizes the need to concurrently explore alternative stem cell sources that do not confer the disadvantages of pluripotency but are widely multipotent so as to yield safe desirable tissues for clinical application as soon as possible.