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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., Introduction The heart is the first organ to function in the vertebrate embryo. In mice, the cardiac mesoderm forms a structure called the cardiac crescent at approximately 7.5 dpc; at [...]

The article states pluripotency as one of the major advances in stem-cell research, which will be of use for reprogramming of adult human cells to produce patient-specific therapies.

Male babies who have cloacal exstrophy probably should not be raised as females, according to a study of 16 boys aged 5 to 16 who had this disorder. Although 14 had been raised as a female, eight declared that they were male and all 14 had interests and attitudes that are typically male. Cloacal exstrophy is a birth defect that affects the entire pelvis and people with this disorder have small or absent sex organs.

The forthcoming transition in the focus of stem cell research from basic science in the development of therapies raises important questions of justice. This transition is marked by increasing interest [...]

Imprinting is an epigenetic modification leading to monoallelic expression of some genes, and disrupted imprinting is believed to be a barrier to human stem cell transplantation, based on studies that suggest that epigenetic marks are unstable in mouse embryonic germ (EG) and embryonic stem (ES) cells. However, stem cell imprinting has not previously been examined directly in humans. We found that three imprinted genes, TSSC5, H19, and SNRPN, show monoallelic expression in in vitro differentiated human EG-derived cells, and a fourth gene, IGF2, shows partially relaxed imprinting at a ratio from 4:1 to 5:1, comparable to that found in normal somatic cells. In addition, we found normal methylation of an imprinting control region (ICR) that regulates H19 and IGF2 imprinting, suggesting that imprinting may not be a significant epigenetic barrier to human EG cell transplantation. Finally, we were able to construct an in vitro mouse model of genomic imprinting, by generating EG cells from 8.5-day embryos of an interspecific cross, in which undifferentiated cells show biallelic expression and acquire preferential parental allele expression after differentiation. This model should allow experimental manipulation of epigenetic modifications of cultured EG cells that may not be possible in human stem cell studies.

Author(s): Peter J. Donovan [1]; John Gearhart [2] The new millennium promised to usher in the era of the human genome. So far, a different area of biology -- stem [...]

Human pluripotent stem cells would be invaluable for in vitro studies of aspects of human embryogenesis. With the goal of establishing pluripotent stem cell lines, gonadal ridges and mesenteries containing primordial germ cells (PGCs, 5-9 weeks post-fertilization) were cultured on mouse STO fibroblast feeder layers in the presence of human recombinant leukemia inhibitory factor, human recombinant basic fibroblast growth factor, and forskolin. Initially, single PGCs in culture were visualized by alkaline phosphatase activity staining, Over a period of 7-21 days, PGCs gave rise to large multicellular colonies resembling those of mouse pluripotent stem cells termed embryonic stem and embryonic germ (EG) cells. Throughout the culture period most cells within the colonies continued to be alkaline phosphatase-positive and tested positive against a panel of five immunological markers (SSEA-1, SSEA-3, SSEA-4, TRA-1-60, and TRA-1-81) that have been used routinely to characterize embryonic stem and EG cells. The cultured cells have been continuously passaged and found to be karyotypically normal and stable. Both XX and XY cell cultures have been obtained. Immunohistochemical analysis of embryoid bodies-collected from these cultures revealed a wide variety of differentiated cell types, including derivatives of all three embryonic germ layers. based on their origin and demonstrated properties, these human PGC-derived cultures meet the criteria for pluripotent stem cells and most closely resemble EG cells.

Hypoxia-inducible factor 1 (HIF-1) is a major regulator of developmental and cellular O2 homeostasis. The factor is induced as a response to physiologically relevant levels of hypoxia, which is an important physiological and developmental stimulus. mRNAs encoding glucose transporters and glycolytic enzymes were found reduced in embryonic cells with HIF-1x deficiency.

A study of hippocampal neurons in transgenic mice indicates the occurrence of both astrocytosis and neurite multiplication with increased levels of the calcium-bonding protein S100-beta. The transgenic mice act as models for studies of the role of S100-beta in glial-neuronal reactions in normal development and the working of the brain. This indicates the importance of increased S100-beta levels in Down syndrome and Alzheimer disease.

A minigene was constructed and transfected into mice and 3T3-L1 preadipocytes to locate the regulatory sites required for expression of the insulin-responsive glucose transporter (GLUT4) gene in adipocytes. The minigene contains substantial 5' and 3' flanking sequence of the GLUT4 gene and a segment of foreign DNA for transcript detection. The results show that regulatory elements for tissue-specific expression and for differentiation-induced expression and cAMP-induced repression of transcription are located within the 14-kb minigene.

Urolithiasis (presence of stones in the urinary tract) occurs as frequently as 1 in 1,000 American children. The incidence of stones is higher among Southeast Asian children, probably due to diets high in carbohydrates and low in purines, while the mechanisms underlying stones in American children are unclear. However, urinary stones are associated with urinary tract abnormalities, infection, metabolic disease, and immobilization (lack of movement by the child). To better understand urolithiasis in children, the cases of 54 patients (22 female), ranging from newborn to 17 years of age, were studied. Symptoms varied from abdominal pain to obviously bloody urine or nausea; symptoms were absent in some cases. Diet or medication may have contributed to stone formation in nine children. High calcium levels in blood or urine occurred in eight children, two of whom had tumors in the parathyroid gland (the gland producing a calcium-regulating hormone), three of whom had been chronically immobilized, one of whom had altered protein metabolism, and three of whom had defective calcium handling. Calcium oxalate stones were most common, occurring in 17 children, while 10 children (9 of whom had previously had urinary tract infections) had stones containing magnesium ammonium phosphate. Urinary tract deformities were present in 19 patients. A total of six children with urolithiasis had been chronically immobilized to allow bone healing following auto accidents or correction of bone deformities. Almost half (25) of the patients had urinary tract infections, of whom all but one had an associated condition such as prior urinary tract surgery. Twenty-one children spontaneously passed the stones, 24 had stones removed by eight different types of surgery, and four had their stones fragmented by extracorporeal shock-wave lithotripsy (crushing of stones by shock waves applied outside the body). Of 50 patients who had follow-up examinations, 11 experienced recurrence of stones, most of whom had urinary tract deformities or prior surgery. The study suggests that existing metabolic or anatomic abnormalities should be identified in children with urolithiasis in order to limit long-term problems. (Consumer Summary produced by Reliance Medical Information, Inc.)

Human pluripotent stem cells (hPSCs) have been derived from the inner cell mass cells of blastocysts (embryonic stem cells) and primordial germ cells of the developing gonadal ridge (embryonic germ cells). Like their mouse counterparts, hPSCs can be maintained in culture in an undifferentiated state and, upon differentiation, generate a wide variety of cell types. Embryoid body (EB) formation is a requisite step in the process of in vitro differentiation of these stem cells and has been used to derive neurons and glia, vascular endothelium, hematopoietic cells, cardiomyocytes, and glucose-responsive insulin-producing cells from mouse PSCs. EBs generated from human embryonic germ cell cultures have also been found to contain a wide variety of cell types, including neural cells, vascular endothelium, muscle cells, and endodermal derivatives. Here, we report the isolation and culture of cells from human EBs as well as a characterization of their gene expression during growth in several different culture environments. These heterogeneous cell cultures are capable of robust and long-term [[is greater than] 70 population doublings (PD)] proliferation in culture, have normal karyotypes, and can be cryopreserved, clonally isolated, and stably transfected. Cell cultures and clonal lines retain a broad pattern of gene expression including simultaneous expression of markers normally associated with cells of neural, vascular/hematopoietic, muscle, and endoderm lineages. The growth and expression characteristics of these EB-derived cells suggest that they are relatively uncommitted precursor or progenitor cells. EB-derived cells may be suited to studies of human cell differentiation and may play a role in future transplantation therapies.

Background: Continence in the bladder exstrophy population requires the coordinated and staged surgical management involving the successful closure of the exstrophy bladder, early epispadias repair, and bladder neck reconstruction (BNR) after an appropriate bladder capacity. In some select cases, initial bladder closure and epispadias may be closed concomitantly. To further delineate factors that may predict eventual continence after bladder neck plasty, we have retrospectively reviewed our patient records. Methods: The records of 88 patients who underwent total bladder exstrophy reconstruction at our institution between 1975 and 1997 were reviewed. 57 patients with BNR were available for analysis after excluding 23 patients awaiting BNR, 3 patients who achieved continence without BNR, 4 patients with recent BNR, but less than one year follow-up, and 1 patients lost to follow-up. Their medical records were reviewed and data analyzed. Results: The median and mean age for primary closure were 10 days and five months, respectively. The average age of BNR was 4.4 years with a mean capacity of 88 cc. Of these 57 patients, 42 (74%) are continent and voiding urethrally without need for augmentation or CIC. 9 (16%) patients have social continence, dry for more than three hours during the day, 2 patients required diversion for continence after failed BNR. 3 patients are wet. Analysis of bladder capacity measurements prior to BNR revealed patients with bladder capacity greater than 75 cc at the time of BNR or with an average increase in capacity of more than 21 cc/year resulted in better outcome. Whereas 2 patients were wet and 2 required urinary diversion in the group with the smaller bladder capacity and poor bladder growth, only one patient was completely wet in the optimal group. No correlation between age of BNR and continence was found. Conclusion: Determinates of continence in the bladder exstrophy population are multifactorial. In our experience 74% are all completely dry, day and night, and 89% can obtain "social continence," being dry for at least 3 hours. However, with careful evaluation of bladder capacity and bladder growth, urinary continence may be improved in this population with better patient selection., David Y. Chan, MD, Robert D. Jeffs, MD, FAAP, and John P. Gearhart, MD, FAAP. James Buchanan Brady Urological Institute, Johns Hopkins Medical Institutions, Baltimore, MD. (Presented by Dr. [...]

Background: The surgical approach to the small newborn exstrophied bladder remains undetermined. Various approaches to long term management of these children have been implemented. The authors investigated their experience in late primary closure of the small exstrophied bladder template. Methods: The charts of 605 patients treated and followed for the exstrophy-epispadias complex were reviewed. Fifty of these underwent primary closure (either at our institution or else. where) after the age of six months. Twenty of these patients had a bladder template which was judged too small to close in the newborn period. The other 30 patients had delayed closure for a variety of financial, geographic, other circumstantial or unknown reasons. Results: Of the 20 children who had delayed closure due to a small bladder template there were 18 males and 2 females. Follow-up time ranged from 1-32 years (mean 21 years). Primary closure was Performed at a mean age of 1.2 years (range 6mo-2yrs). Osteotomies were performed in 17. Nine of 20 patients achieved continence after gaining sufficient bladder capacity for bladder neck reconstruction. Five of 20 patients required enterocystoplasty to augment bladder volume, and Perform clean intermittent catheterization (2 per stoma, and 3 per urethra). One patient required a colon conduit for very small bladder and one a cystectomy and ureterosigmoidostomy due to rhabdomyosarcoma. Four patients are incontinent, 2 of these are awaiting bladder neck reconstruction, and 2 had a failed bladder neck reconstruction. Conclusion: Delayed primary closure of a small exstrophied bladder template permits the native bladder tissue time to grow to a size that is feasible for successful closure in most patients. An epispadias repair can usually be performed at the same time especially with testosterone stimulation. Bladder neck reconstructive techniques have achieved continence without the need for augmentation or bladder replacement in 45% of patients. Even in patients who do not achieve adequate capacity for bladder neck reconstruction, preservation of the native bladder template allows for easier future augmentation and ureteral reimplantation., Jennifer Dodson, MD, Ilhami Surer, MD, Linda Baker, MD and John P. Gearhart, MD, FAAP. Division of Pediatric Urology, Brady Urological Institute, The Johns Hopkins Hospital, Baltimore, [...]

Background: In the exstrophy-epispadias population, male adolescents consider the oddly appearing genitalia, with a short widened penis, to be a greater psychosocial problem than incontinence and therefore every effort should be made to restore the penis to a normal status. Therefore the authors evaluated their ten year experience with the modified Cantwell-Ransley epispadias repair technique to determine the complications and long term results. Methods: A retrospective chart review was performed on 605 exstrophy-epispadias data-base patients. 93 males (79 with classic bladder exstrophy and 14 with complete epispadias) underwent a modified Cantwell-Ransley epispadias repair over the last ten years. Primary repair was performed in 77 boys (65 with classic bladder exstrophy and 12 with epispadias), and secondary repair was performed after prior failed reconstruction in 16 boys (14 classic exstrophy and 2 with complete epispadias). Epispadias repair was combined with reclosure of bladder exstrophy in 10 patients. Results: At a mean follow up of 68 months (4-120mo), 87 patients had a horizontal or downward angled penis while standing and 88 patients pass urine per urethra. Fistulas developed in 22 patients (23%). In 4 patients the fistulas closed spontaneously within the first 3 months after surgery and 18 patients (19%) underwent a separate procedure to close the urethrocutaneous fistula. Seven patients developed a urethral stricture at the proximal anastomotic area and 5 patients had minor skin separations (4 exstrophy, 1 epispadias) of the dorsal penile skin closure. Catheterization or cystoscopy in 77 children revealed an easily negotiable neourethral channel. Conclusion: The authors conclude from their ten year follow-up that the single stage modified Cantwell-Ransley epispadias repair technique creates a cosmetically satisfactory penis and produces a straight, catheterizable neourethral channel with an acceptable complication rate. However, definitive assessment of genital reconstruction must be deferred until all of these boys are sexually mature and active. While many methods of epispadias repair exist, meticulous follow-up of the urethra, patient selection and surgical experience remain the milestones for success., Ilhami Surer, MD, Linda A. Baker, MD, Robert D. Jeffs, MD, FAAP and John P. Gearhart, MD, FAAP. Division of Pediatric Urology, Brady Urological Institute, The Johns Hopkins Hospital and [...]

Background: We analyzed shape and stress on the hip joint in exstrophy and reviewed the status of hips in adults with uncorrected exstrophy. Methods: Fourteen patients aged 16 to 52 years born with classic bladder exstrophy were studied. Patients were matched for age and gender with 14 controls. AP pelvis radiographs were used for biomechanical analysis based on the single-stance phase of gait. Joint force and joint stress (force/area) were calculated relative to partial body weight by the method of Legal (1987). Clinical assessment was by Iowa hip score and radiographic grading. Statistical analysis was done using t tests. Results: For the exstrophy patients, the mean relative joint force was significantly higher than control (4.2 [+ or -] 0.91; 3.0 [+ or -] 0.3) (p [is less than] 0.01). The mean relative joint stress for exstrophy patients was significantly higher as well (p [is less than] 0.05). The increase in force and stress appeared to be due to three factors: (1) the mean distance from the body center to the center of the femoral head was significantly increased (p [is less than] 0.001) in bladder exstrophy (12.35 [+ or -] 1.05 cm) versus controls (10.31 [+ or -] 0.70 cm) (approximately 30% increased). (2) The mean distance from the greater trochanter to the femoral head center (f) was significantly less (p [is less than] 0.02) for exstrophy patients (5.2 [+ or -] 1.2 cm) compared to controls (6.5 [+ or -] 0.8 cm). (3) The center-edge angle (CO angle) was significantly decreased in exstrophy patients (25.0 [+ or -] 9 deg.) Versus control (33 [+ or -] deg) (p [is less than] 0.05). Two of the adults with exstrophy had decreased Iowa hip scores and radiographic evidence of arthrosis. Conclusions: The force and stress on the hip joint are increased in adult bladder exstrophy patients. Increased joint load and stress can lead to an earlier onset of DJD. These results need corroboration by larger series. It remains to be seen whether closing the pelvic ring at exstrophy closure may help reduce these factors and affect the outcome of the hip., M.M. Jani, BA, Paul D. Sponseller, M.D., FAAP, John P. Gearhart, M.D., FAAP. Johns Hopkins Hospital, Baltimore [...]

Background: While some case series have suggested delayed linear growth after enterocystoplasty, none of these were case-controlled and the series did not necessarily include patients whose bladders were augmented for identical reasons. Therefore, the present study was performed to examine the hypothesis that intestinal bladder augmentation is associated with reduced linear growth. Methods: Fifty patients who had undergone bladder augmentation for incontinence resulting from bladder exstrophy were randomly selected from our institution's patient data-base and matched for gender, age and type of exstrophy with 50 non-augmented bladder exstrophy patients. Patients were then contacted and asked to permit their pediatricians to release their growth charts. Once consent was obtained, the charts were requested from the pediatricians. Evaluable data (defined as at least one height pre and post augmentation) were obtained for 20 (40%) augmented and 14 (28%) non augmented patients. Results: The mean age at operation was 7.7 years. Delayed growth defined by a postoperative drop in percentile height occurred in 18 (90%) of augmented patients, with a mean loss of 17 percentile points. In the control group, delayed growth after 7.7 years of age occurred in 4 (29%) patients, with the control group as a whole gaining average of 8 percentile points. A Shapiro-Wilk test found the heights to be normally distributed (p=0.19 and 0.93 respectively) and a t-test showed the mean height change to be significantly different between the two groups (p=0.006). The average follow up periods of 6 and 7.4 years in the augmented and non-augmented groups, respectively, were not significantly different (p=0.19). Conclusion: Intestinal bladder augmentation is associated with a nearly universal decrease in percentile height. Analysis of subtle metabolic alterations may provide information to help minimize or prevent growth impediment in the future. Alternatives to intestinal augmentation should continue to be pursued., Jennifer L. Dodson, MD, David-Alexandre C. Gros, MD, Uri A. Lopatin, BA, John P. Gearhart, MD, FAAP, Richard I. Silver MD, Steven G. Docimo, MD, FAAP. Division of Pediatric Urology, [...]

Background: To assess results, applications, and complications of pelvic osteotomy producing continence in patients with the exstrophy/epispadias complex. Methods: Eighty-five patients who underwent pelvic osteotomy and external fixation were reviewed at a minimum of two year follow up (mean 4.8 years). Seventy-two patients had classic bladder exstrophy and 13 patients had cloacal exstrophy. Indications for osteotomy were to achieve a tension-free closure of the bladder and lower abdominal wall and/or to approximate pelvic floor muscles at the time of later bladder neck reconstruction. The patients were stratified into five different groups of age at surgery for analysis. Of these patients, 37 had anterior innominate osteotomy, 40 combined anterior innominate and posterior iliac osteotomy, 6 posterior lilac osteotomy, and 2 suprapublic ramotomy osteotomies were performed. Results: The mean age at surgery was 2.9 [+ or -] 3.7 years (range 3 days to 13 years old). Osteotomy was performed at the time of initial bladder closure in 21, reclosure in 34, and bladder neck reconstruction in 30. In classic exstrophy patients, diastasis was corrected to an initial mean of 2.8 cm and a final mean of 3.8 cm. Cloacal exstrophy patients had significantly greater initial and residual diastasis. Maintenance of diastasis correction increased continuously with age at surgery. Wound dehiscence or bladder prolapse occurred in 4% of patients after primary closures and 0% after reclosure. Daytime continence was achieved in 75% of patients. The degree of continence was not correlated with percent correction of diastasis. Complications include transient femoral nerve palsy in 7 cases (8%) which all resolved spontaneously after three months and were probably due to tension on the inguinal ligament; delayed union in 3; late pin-track osteomyelitis in one. Conclusion: Pelvic osteotomy is useful in helping achieve two goals of exstrophy treatment: successful bladder and abdominal wall closure along with urinary continence. The authors prefer an anterior approach because of the single stage positioning and accuracy of fixator application. Long-term maintenance of diastasis is least in the younger patients, probably because of continued undergrowth of the anterior pelvic segment. The authors prefer closure without osteotomy in young infants if this can be achieved early on without tension. However, in other cases, pelvic osteotomies are affective in achieving treatment aims with an acceptably low complication rate., Paul D. Sponseller, M.D., FAAP, M.M. Jani, BA, and John P. Gearhart, M.D., FAAP. Johns Hopkins Hospital, Baltimore, [...]

Embryonic stem (ES) cells--pluripotential stem cells that give rise to all adult cell types--can be derived from the blastocyst, a preimplantation stage embryo (1), or from primordial germ cells, cells [...]

If human neural stem cells were implanted into the brains of other primates what might this do to the mind of the recipient? Could such grafting teach us anything of [...]

THE RECENT TRIAL IN THE PRESS OF THE ETHICS and scientific validity of publications on human somatic cell nuclear transfer ('Evidence of a pluripotent human embryonic stem cell line derived [...]

Objectives. To identify and study adults (21 years or older) who have a 46,XY karyotype and presented as infants or children with genital ambiguity, including a small phallus and perineoscrotal hypospadias, reared male or female. Methods. Participants were classified according to the cause underlying their intersex condition based on review of medical and surgical records. Long-term medical and surgical outcome was assessed with a written questionnaire and physical examination. Long-term psychosexual development was assessed with a written questionnaire and semistructured interview. Results. Thirty-nine (72%) of 54 eligible patients participated. The cause underlying genital ambiguity of participants included partial androgen insensitivity syndrome (n = 14; 5 men and 9 women), partial gonadal dysgenesis (n = 11; 7 men and 4 women), and other intersex conditions. Men had significantly more genital surgeries (mean: 5.8) than women (mean: 2.1), and physician-rated cosmetic appearance of the genitalia was significantly worse for men than for women. The majority of participants were satisfied with their body image, and men and women did not differ on this measure. Most men (90%) and women (83%) had sexual experience with a partner. Men and women did not differ in their satisfaction with their sexual function. The majority of participants were exclusively heterosexual, and men considered themselves to be masculine and women considered themselves to be feminine. Finally, 23% of participants (5 men and 4 women) were dissatisfied with their sex of rearing determined by their parents and physicians. Conclusions. Either male or female sex of rearing can lead to successful long-term outcome for the majority of cases of severe genital ambiguity in 46,XY individuals. We discuss factors that should be considered by parents and physicians when deciding on a sex of rearing for such infants. Pediatrics 2002;110(3). URL: http://www. pediatrics.org/cgi/content/full/110/3/e31; intersex, sex assignment, gender, androgen insensitivity, gonadal dysgenesis, psychosexual, genital reconstruction, hormone replacement.

Pluripotential stem cells, present in the early stages of embryo development, can generate all of the cell types in a fetus and in the adult and are capable of self-renewal. [...]