Christine Hill

Expression of pluripotent stem cell markers in the human fetal testis.

Human primordial germ cells (PGCs) have proven to be a source of pluripotent stem cells called embryonic germ cells (EGCs). However, the developmental potency of these cells in the fetal gonad still remains elusive. Thus, this study provides a comprehensive analysis of pluripotent and germ cell marker expression in human fetal testis 7–15 weeks postfertilization (pF) and compares this expression to their ability to derive EGCs. Although the majority of germ cells expressed stem cell markers stage‐specific embryonic antigen (SSEA) 1, SSEA4, EMA‐1, and alkaline phosphatase, only a small percentage of those (<1%) expressed OCT4, CKIT, and NANOG. Specifically, the number of OCT4+/CKIT+/NANOG+ cells significantly increased in the developing cords during weeks 7–9, followed by a gradual decline into week 15 pF. By week 15 pF, the remaining OCT4+/CKIT+/NANOG+ cells were found in the cords surrounding the periphery of the testis, and the predominant germ cells, CKIT+ cells, no longer expressed OCT4 or NANOG. Based on morphology and early germ cell marker expression, including VASA, PUM2, and DAZL, we suggest these cells are mitotically active gonocytes or prespermatogonia. Importantly, the number of OCT4+ cells correlated with an increase in the number of EGC colonies derived in culture. Interestingly, two pluripotent markers, Tra‐1–60 and Tra‐1–81, although highly expressed in EGCs, were not expressed by PGCs in the gonad. Together, these results suggest that PGCs maintain expression of pluripotent stem cell markers during and after sexual differentiation of the gonad, albeit in very low numbers.

Mouse ES cell–derived cardiac precursor cells are multipotent and facilitate identification of novel cardiac genes

Although the differentiation of ES cells to cardiomyocytes has been firmly established, the extent to which corresponding cardiac precursor cells can contribute to other cardiac populations remains unclear. To determine the molecular and cellular characteristics of cardiac-fated populations derived from mouse ES (mES) cells, we isolated cardiac progenitor cells (CPCs) from differentiating mES cell cultures by using a reporter cell line that expresses GFP under the control of a cardiac-specific enhancer element of Nkx2-5, a transcription factor expressed early in cardiac development. This ES cell-derived CPC population initially expressed genetic markers of both stem cells and mesoderm, while differentiated CPCs displayed markers of 3 distinct cell lineages (cardiomyocytes, vascular smooth muscle cells, and endothelial cells)--Flk1 (also known as Kdr), c-Kit, and Nkx2-5, but not Brachyury--and subsequently expressed Isl1. Clonally derived CPCs also demonstrated this multipotent phenotype. By transcription profiling of CPCs, we found that mES cell-derived CPCs displayed a transcriptional signature that paralleled in vivo cardiac development. Additionally, these studies suggested the involvement of genes that we believe were previously unknown to play a role in cardiac development. Taken together, our data demonstrate that ES cell-derived CPCs comprise a multipotent precursor population capable of populating multiple cardiac lineages and suggest that ES cell differentiation is a valid model for studying development of multiple cardiac-fated tissues.

Expression of pluripotent stem cell markers in the human fetal ovary

BACKGROUND Human primordial germ cells (PGCs) can give rise to pluripotent stem cells such as embryonal carcinoma cells (ECCs) and embryonic germ cells (EGCs). METHODS In order to determine whether PGCs express markers associated with pluripotency in EGCs and ECCs, the following study cross examines the expression patterns of multiple pluripotent markers in the human fetal ovary, 5.5-15 weeks post-fertilizaton (pF) and relates this expression with the ability to derive pluripotent EGCs in vitro. RESULTS Specific subpopulations were identified which included OCT4(+)/Nanog(+)/cKIT(+)/VASA(+) PGCs and oogonia. Interestingly, these cells also expressed SSEA1 and alkaline phosphatase (AP) and SSEA4 expression occurred throughout the entire gonad. Isolation of SSEA1(+) cells from the gonad resulted in AP(+) EGC colony formation. The number of OCT4(+) or Nanog(+) expressing cells peaked by week 8 and then diminished after week 9 pF, as oogonia enter meiosis. In addition, the efficiency of EGC derivation was associated with the number of OCT4(+) cells. TRA-1-60 and TRA-1-81 were only detected in the lining of the mesonephric ducts and occasionally in the gonad. CONCLUSIONS These results demonstrate that PGCs, a unipotent cell, express most, but not all, of the markers associated with pluripotent cells in the human fetal ovary.

Implantation of Mouse Embryonic Stem Cell-Derived Cardiac Progenitor Cells Preserves Function of Infarcted Murine Hearts

Stem cell transplantation holds great promise for the treatment of myocardial infarction injury. We recently described the embryonic stem cell-derived cardiac progenitor cells (CPCs) capable of differentiating into cardiomyocytes, vascular endothelium, and smooth muscle. In this study, we hypothesized that transplanted CPCs will preserve function of the infarcted heart by participating in both muscle replacement and neovascularization. Differentiated CPCs formed functional electromechanical junctions with cardiomyocytes in vitro and conducted action potentials over cm-scale distances. When transplanted into infarcted mouse hearts, CPCs engrafted long-term in the infarct zone and surrounding myocardium without causing teratomas or arrhythmias. The grafted cells differentiated into cross-striated cardiomyocytes forming gap junctions with the host cells, while also contributing to neovascularization. Serial echocardiography and pressure-volume catheterization demonstrated attenuated ventricular dilatation and preserved left ventricular fractional shortening, systolic and diastolic function. Our results demonstrate that CPCs can engraft, differentiate, and preserve the functional output of the infarcted heart.

Efficient differentiation of human embryonic stem cells into oligodendrocyte progenitors for application in a rat contusion model of spinal cord injury

ABSTRACT This study utilized a contusion model of spinal cord injury (SCI) in rats using the standardized NYU-MASCIS impactor, after which oligodendrocyte progenitor cells (OPCs) derived from human embryonic stem cell (ESC) were transplanted into the spinal cord to study their survival and migration route toward the areas of injury. One critical aspect of successful cell-based SCI therapy is the time of injection following injury. OPCs were injected at two clinically relevant times when most damage occurs to the surrounding tissue, 3 and 24 hours following injury. Migration and survivability after eight days was measured postmortem. In-vitro immunofluorescence revealed that most ESC-derived OPCs expressed oligodendrocyte markers, including CNPase, GalC, Olig1, O4, and O1. Results showed that OPCs survived when injected at the center of injury and migrated away from the injection sites after one week. Histological sections revealed integration of ESC-derived OPCs into the spinal cord with contusion injury without disruption to the parenchyma. Cells survived for a minimum of eight days after injury, without tumor or cyst formation. The extent of injury and effect of early cell transplant was measured using behavioral and electrophysiological assessments which demonstrated increased neurological responses in rats transplanted with OPCs compared to controls.

Intratesticular Androgen Levels, Androgen Receptor Localization, and Androgen Receptor Expression in Adult Rat Sertoli Cells

Abstract In the rat, quantitatively normal spermatogenesis is maintained only when intratesticular testosterone (ITT) levels greatly exceed the peripheral T concentration. When ITT concentrations fall below a threshold, germ cells are lost at specific stages of the seminiferous cycle. Germ cells can be restored by high doses of T that binds to androgen receptors (AR) in Sertoli cells. However, the relationships between germ cell dynamics, AR-mediated molecular events, and ITT concentrations are not established. ITT levels may regulate germ cell life and death through an effect on AR localization and AR mRNA or protein levels within Sertoli cells at specific stages of the cycle. We determined AR localization and mRNA and protein expression in adult rat Sertoli cells in relation to reduced and then restored ITT concentrations in vivo. ITT levels were reduced by implanting rats with T- and estradiol (E)-filled capsules for 7–28 days and subsequently restored with large T-filled capsules. AR is normally localized within Sertoli cell nuclei at stages VII–VIII of the seminiferous epithelium. After T/E treatment, AR immunostaining in Sertoli cell nuclei became nondetectable by 14–28 days but was restored 6 h following T restoration. The loss of Sertoli cell nuclear AR localization correlated with increasing numbers of apoptotic germ cells. AR mRNA levels in isolated Sertoli cells did not change through 14 days of T/E treatment, increased significantly by Day 28, and remained elevated 24 h after T restoration. AR mRNA levels in microdissected tubules at stages II–IV, VI–VIII, and IX–XII did not decrease through 14 days of T/E treatment. In contrast, AR protein levels were reduced in seminiferous tubules by Day 14 and in testes at Day 28 post-T/E treatment but were restored within 24 h by T repletion. Therefore, the reduction of ITT concentration results in a time-dependent redistribution of AR and reduced AR protein but not AR mRNA levels in Sertoli cells. Repletion of T restored AR protein and it relocated to Sertoli cell nuclei. By an unknown mechanism, T regulates AR localization within Sertoli cells to determine germ cell life or death.

Phosphorylation of Mitogen‐Activated Protein Kinase 8 (MAPK8) Is Associated With Germ Cell Apoptosis and Redistribution of the Bcl2‐Modifying Factor (BMF)

Successful spermatogenesis requires that germ cells remain in physical contact with Sertoli cells until spermiation. Previous studies have shown that the Bcl2-modifying factor (BMF) is a proapoptotic protein found in many epithelial cells which, when phosphorylated by the active form of mitogen-activated protein kinase 8 (p-MAPK8), initiates apoptosis in response to loss of adhesion of the cells to their basal lamina. Based on this, we hypothesized that p-MAPK8 and BMF may play important roles in the apoptotic death of testicular germ cells in response to their detachment from Sertoli cells. Immunohistochemical analysis of the normal rat testis revealed p-MAPK8 expression in spermatocytes and elongated spermatids but not in round spermatids. This localization was opposite to that of BMF, which is expressed in round spermatids but not in spermatocytes or elongated spermatids. When freshly isolated germ cells were cultured in the absence of Sertoli cells, a condition in which there was widespread germ cell apoptosis, an increase in p-MAPK8 relative to overall MAPK8 protein, was seen by Western blot analysis. Additionally, immunocytochemical analysis showed an increase in immunoreactive p-MAPK8 in round spermatids and spermatocytes in association with BMF expression. From these correlative data, we propose that the activation of MAPK8 and redistribution of BMF may be integrally involved in the mechanism by which specific germ cells undergo programmed cell death in response to their detachment from Sertoli cells.

Cells derived from human umbilical cord blood support the long-term growth of undifferentiated human embryonic stem cells.

Various types of human cells have been tested as feeder cells for the undifferentiated growth of human embryonic stem cells (hESCs) in vitro. We report here the successful culture of two hESC lines (H1 and H9) on human umbilical cord blood (UCB)-derived fibroblast-like cells. These cells permit the long-term continuous growth of undifferentiated and pluripotent hESCs. The cultured hESCs had normal karyotypes, expressed OCT-4, SSEA-4, TRA-1-60, and TRA-1-81, formed cystic embryonic body in vitro and teratomas in vivo after injected into immunodeficient mice. The wide availability of clinical-grade human UCB makes it a promising source of support cells for the growth of hESC for use in cell therapies.

Bariatric endoscopy: State-of-the-art

Purpose of review This review aims to present the current state of the field, highlight recent developments, and describe the clinical outcomes of these endoscopic bariatric and metabolic procedures. Recent findings There are an increasing number of devices and techniques that are available for the endoluminal treatment of obesity. It is now possible to offer FDA approved endoscopic therapies for patients with BMI as high as 55 kg/m2. Although tremendous advances have been made, there is room for additional emphasis with regards to the metabolic improvements seen with these therapies, in addition to highlighting weight loss outcomes. Furthermore, for optimal outcomes, it is imperative that all endoluminal techniques are combined with at least a moderate intensity diet and lifestyle intervention for a minimum of 12 months. Summary This field bridges a gap between lifestyle counseling with or without pharmaceutical treatment and the most effective treatment of obesity, bariatric surgery. Owing to its minimally invasive nature, endoscopic metabolic and bariatric therapy has the potential to appeal patients who either do not qualify or do not want bariatric surgery and have not been adequately treated with medical therapy. In addition, these therapies may be beneficial early on in the onset of obesity.

Endoluminal weight loss and metabolic therapies: current and future techniques

Obesity is a public health epidemic associated with a number of comorbidities, most notably type 2 diabetes and hypertension, as well as elevated all‐cause mortality. The treatment for obesity and its associated comorbidities has most recently expanded into the field of bariatric endoscopy. This field bridges a gap between lifestyle counseling with or without pharmaceutical treatment and the most effective treatment of obesity, bariatric surgery. Because of its minimally invasive nature, bariatric endoscopic therapy has the potential to appeal to the large sector of the obese population that resists surgery, as well as those early in the onset of obesity. To date, five endoscopic devices have been approved by the U.S. Food and Drug Administration for the treatment of obesity, and many more are in development, undergoing clinical trials, or being used around the world. Here, we present the current state of the field, highlight recent developments, and describe the clinical outcomes of these minimally invasive procedures in terms of weight loss, improvement in metabolic profile, and reduction in comorbidities.