Renee Reijo-Pera

Isolation and Characterization of Pluripotent Human Spermatogonial Stem Cell-Derived Cells

Several reports have documented the derivation of pluripotent cells (multipotent germline stem cells) from spermatogonial stem cells obtained from the adult mouse testis. These spermatogonia‐derived stem cells express embryonic stem cell markers and differentiate to the three primary germ layers, as well as the germline. Data indicate that derivation may involve reprogramming of endogenous spermatogonia in culture. Here, we report the derivation of human multipotent germline stem cells (hMGSCs) from a testis biopsy. The cells express distinct markers of pluripotency, form embryoid bodies that contain derivatives of all three germ layers, maintain a normal XY karyotype, are hypomethylated at the H19 locus, and express high levels of telomerase. Teratoma assays indicate the presence of human cells 8 weeks post‐transplantation but limited teratoma formation. Thus, these data suggest the potential to derive pluripotent cells from human testis biopsies but indicate a need for novel strategies to optimize hMGSC culture conditions and reprogramming. STEM CELLS 2009;27:138–149

Endothelial Cells Derived From Human iPSCS Increase Capillary Density and Improve Perfusion in A Mouse Model of Peripheral Arterial Disease

Objective—Stem cell therapy for angiogenesis and vascular regeneration has been investigated using adult or embryonic stem cells. In the present study, we investigated the potential of endothelial cells (ECs) derived from human induced pluripotent stem cells (hiPSCs) to promote the perfusion of ischemic tissue in a murine model of peripheral arterial disease. Methods and Results—Endothelial differentiation was initiated by culturing hiPSCs for 14 days in differentiation media supplemented with BMP-4 and vascular endothelial growth factor. The hiPSC-ECs exhibited endothelial characteristics by forming capillary-like structures in matrigel and incorporating acetylated-LDL. They stained positively for EC markers such as KDR, CD31, CD144, and eNOS. In vitro exposure of hiPSC-ECs to hypoxia resulted in increased expression of various angiogenic related cytokines and growth factors. hiPSC-ECs were stably transduced with a double fusion construct encoded by the ubiquitin promoter, firefly luciferase for bioluminescence imaging and green fluorescence protein for fluorescent detection. The hiPSC-ECs (5×105) were delivered by intramuscular injection into the ischemic hindlimb of SCID mice at day 0 and again on day 7 after femoral artery ligation (n=8). Bioluminescence imaging showed that hiPSC-ECs survived in the ischemic limb for at least 2 weeks. In addition, laser Doppler imaging showed that the ratio of blood perfusion was increased by hiPSC-EC treatment by comparison to the saline-treated group (0.58±0.12 versus 0.44±0.04; P=0.005). The total number of capillaries in the ischemic limb of mice receiving hiPSC-EC injections was greater than those in the saline-treated group (1284±155 versus 797±206 capillaries/mm2) (P<0.002). Conclusion—This study is a first step toward development of a regenerative strategy for peripheral arterial disease based on the use of ECs derived from hiPSCs.

Characterization of the human ESC transcriptome by hybrid sequencing

Significance Isoform identification and discovery are an important goal for transcriptome analysis because the majority of human genes express multiple isoforms with context- and tissue-specific functions. Better annotation of isoforms will also benefit downstream analysis such as expression quantification. Current RNA-Seq methods based on short-read sequencing are not reliable for isoform discovery. In this study we developed a new method based on the combined analysis of short reads and long reads generated, respectively, by second- and third-generation sequencing and applied this method to obtain a comprehensive characterization of the transcriptome of the human embryonic stem cell. The results showed that large gain in sensitivity and specificity can be achieved with this strategy. Although transcriptional and posttranscriptional events are detected in RNA-Seq data from second-generation sequencing, full-length mRNA isoforms are not captured. On the other hand, third-generation sequencing, which yields much longer reads, has current limitations of lower raw accuracy and throughput. Here, we combine second-generation sequencing and third-generation sequencing with a custom-designed method for isoform identification and quantification to generate a high-confidence isoform dataset for human embryonic stem cells (hESCs). We report 8,084 RefSeq-annotated isoforms detected as full-length and an additional 5,459 isoforms predicted through statistical inference. Over one-third of these are novel isoforms, including 273 RNAs from gene loci that have not previously been identified. Further characterization of the novel loci indicates that a subset is expressed in pluripotent cells but not in diverse fetal and adult tissues; moreover, their reduced expression perturbs the network of pluripotency-associated genes. Results suggest that gene identification, even in well-characterized human cell lines and tissues, is likely far from complete.

A characterization of the relationship of ovarian reserve markers with age

OBJECTIVE To identify markers of ovarian age that best match the pattern of oocyte loss seen in histology specimens. DESIGN Cross-sectional study. SETTING University. PATIENT(S) Caucasian women (n = 252) aged 25-45 years. INTERVENTION(S) none. MAIN OUTCOME MEASURE(S) The relationship between antral follicle count (AFC), antimüllerian hormone (AMH), inhibin B, FSH, and E(2) with age was estimated using the power model, which previously has been shown to most accurately describe oocyte loss in histologic specimens. The power model was fit to each marker and used to compare the rates of change at ages 30 and 40 with the histologic pattern. Among those markers following the pattern, R(2) was used to compare the degree of relationship with age. RESULT(S) Both AMH levels and AFC exhibited significant progressive declines with age. The average rates of loss per year for AFC and AMH were, respectively, -0.57 and -1.09 at age 30, and -1.33 and -3.06 at age 40. FSH, inhibin B, and E(2) did not exhibit progressive rates of change. The R(2) for AFC was 27.3% and for AMH was 22.7%. CONCLUSION(S) Only AFC and AMH follow the pattern of oocyte loss observed histologically. Although AMH may be more cost-effective, AFC is a slightly more accurate noninvasive measure for ovarian aging.

Skeletogenic phenotype of human Marfan embryonic stem cells faithfully phenocopied by patient-specific induced-pluripotent stem cells

Marfan syndrome (MFS) is a heritable connective tissue disorder caused by mutations in the gene coding for FIBRILLIN-1 (FBN1), an extracellular matrix protein. MFS is inherited as an autosomal dominant trait and displays major manifestations in the ocular, skeletal, and cardiovascular systems. Here we report molecular and phenotypic profiles of skeletogenesis in tissues differentiated from human embryonic stem cells and induced pluripotent stem cells that carry a heritable mutation in FBN1. We demonstrate that, as a biological consequence of the activation of TGF-β signaling, osteogenic differentiation of embryonic stem cells with a FBN1 mutation is inhibited; osteogenesis is rescued by inhibition of TGF-β signaling. In contrast, chondrogenesis is not perturbated and occurs in a TGF-β cell-autonomous fashion. Importantly, skeletal phenotypes observed in human embryonic stem cells carrying the monogenic FBN1 mutation (MFS cells) are faithfully phenocopied by cells differentiated from induced pluripotent-stem cells derived independently from MFS patient fibroblasts. Results indicate a unique phenotype uncovered by examination of mutant pluripotent stem cells and further demonstrate the faithful alignment of phenotypes in differentiated cells obtained from both human embryonic stem cells and induced pluripotent-stem cells, providing complementary and powerful tools to gain further insights into human molecular pathogenesis, especially of MFS.

Male Sperm Motility Dictated by Mother's mtDNA

We are grateful to Dr. Paul Turek, Dr. Eugene Xu, and Joyce Tung for their helpful comments and support. F.A.M. is supported by a Ford Foundation predoctoral fellowship. R.A.R.-P. is supported by the Searle Scholarship Foundation and grants from the National Institutes of Health.

Novel missense mutations of the Deleted-in-AZoospermia-Like (DAZL) gene in infertile women and men.

BackgroundThe Deleted-in-AZoospermia-Like (DAZL) gene has homologs required for germ cell development in many organisms. Recently, we showed that there are several common polymorphisms within the DAZL gene that are associated with age at ovarian failure/menopause and sperm count.MethodsHere we sought to identify rare mutations in DAZL and examine their phenotypes in men and women. We sequenced the DAZL gene in 519 individuals; sequences spanned the entire coding region of the gene.ResultsWe report the identification of four putative missense mutations in DAZL. Three individuals that were heterozygous for a DAZL mutation reported having children, while two individuals that were homozygous reported no children. These mutations were found only in infertile men and women.ConclusionGiven the strong data associating DAZL polymorphisms and deletions with fertility in humans and model organisms, we suggest that these mutations may be associated with age at menopause and/or sperm count and warrant further biochemical and genetic investigation.

Vascular Progenitors From Cord Blood–Derived Induced Pluripotent Stem Cells Possess Augmented Capacity for Regenerating Ischemic Retinal Vasculature

Background— The generation of vascular progenitors (VPs) from human induced pluripotent stem cells (hiPSCs) has great potential for treating vascular disorders such as ischemic retinopathies. However, long-term in vivo engraftment of hiPSC-derived VPs into the retina has not yet been reported. This goal may be limited by the low differentiation yield, greater senescence, and poor proliferation of hiPSC-derived vascular cells. To evaluate the potential of hiPSCs for treating ischemic retinopathies, we generated VPs from a repertoire of viral-integrated and nonintegrated fibroblast and cord blood (CB)–derived hiPSC lines and tested their capacity for homing and engrafting into murine retina in an ischemia-reperfusion model. Methods and Results— VPs from human embryonic stem cells and hiPSCs were generated with an optimized vascular differentiation system. Fluorescence-activated cell sorting purification of human embryoid body cells differentially expressing endothelial/pericytic markers identified a CD31+CD146+ VP population with high vascular potency. Episomal CB-induced pluripotent stem cells (iPSCs) generated these VPs with higher efficiencies than fibroblast-iPSC. Moreover, in contrast to fibroblast-iPSC-VPs, CB-iPSC-VPs maintained expression signatures more comparable to human embryonic stem cell VPs, expressed higher levels of immature vascular markers, demonstrated less culture senescence and sensitivity to DNA damage, and possessed fewer transmitted reprogramming errors. Luciferase transgene-marked VPs from human embryonic stem cells, CB-iPSCs, and fibroblast-iPSCs were injected systemically or directly into the vitreous of retinal ischemia-reperfusion–injured adult nonobese diabetic-severe combined immunodeficient mice. Only human embryonic stem cell– and CB-iPSC–derived VPs reliably homed and engrafted into injured retinal capillaries, with incorporation into damaged vessels for up to 45 days. Conclusions— VPs generated from CB-iPSCs possessed augmented capacity to home, integrate into, and repair damaged retinal vasculature.

In vivo molecular MRI of cell survival and teratoma formation following embryonic stem cell transplantation into the injured murine myocardium

Embryonic stem cells (ESCs) have shown the potential to restore cardiac function after myocardial injury. Superparamagnetic iron oxide nanoparticles (SPIO) have been widely employed to label ESCs for cellular MRI. However, nonspecific intracellular accumulation of SPIO limits long‐term in vivo assessment of the transplanted cells. To overcome this limitation, a novel reporter gene (RG) has been developed to express antigens on the ESC surface. By employing SPIO‐conjugated monoclonal antibody against these antigens (SPIO‐MAb), the viability of transplanted ESCs can be detected in vivo. This study aims to develop a new molecular MRI method to assess in vivo ESC viability, proliferation, and teratoma formation. The RG is designed to express 2 antigens (hemagglutinin A and myc) and luciferase on the ESC surface. The two antigens serve as the molecular targets for SPIO‐MAb. The human and mouse ESCs were transduced with the RG (ESC‐RGs) and transplanted into the peri‐infarct area using the murine myocardial injury model. In vivo MRI was performed following serial intravenous administration of SPIO‐MAb. Significant hypointense signal was generated from the viable and proliferating ESCs and subsequent teratoma. This novel molecular MRI technique enabled in vivo detection of early ESC‐derived teratoma formation in the injured murine myocardium. Magn Reson Med, 2011.

Adverse childhood experiences and repeat induced abortion.

OBJECTIVE The objective of the study was to characterize the backgrounds of women who have repeat abortions. STUDY DESIGN In a cross-sectional study of 259 women (mean age, 35.2 ± 5.6 years), the relation between adverse experiences in childhood and risk of having 2 or more abortions vs 0 or 1 abortion was examined. Self-reported adverse events occurring between the ages of 0 and 12 years were summed. RESULTS Independent of confounding factors, women who experienced more abuse, personal safety, and total adverse events in childhood were more likely to have 2 or more abortions vs 0 abortions (odds ratio [OR], 2.56; 95% confidence interval [CI], 1.15-5.71; OR, 2.74; 95% CI, 1.29-5.82; and OR, 1.59; 95% CI, 1.21-2.09, respectively) and vs 1 abortion (OR, 5.83; 95% CI, 1.71-19.89; OR, 2.23; 95% CI, 1.03-4.81; and OR, 1.37; 95% CI, 1.04-1.81, respectively). Women who experienced more family disruption events in childhood were more likely to have 2 or more abortions vs 0 abortions (OR, 1.75; 95% CI, 1.14-2.69) but not vs 1 abortion (OR, 1.16; 95% CI, 0.79-1.70). CONCLUSION Women who have repeat abortions are more likely to have experienced childhood adversity than those having 0 or 1 abortion.