Randall J. Urban

Production of stem cells with embryonic characteristics from human umbilical cord blood

Abstract.  When will embryonic stem cells reach the clinic? The answer is simple – not soon! To produce large quantities of homogeneous tissue for transplantation, without feeder layers, and with the appropriate recipients immunological phenotype, is a significant scientific hindrance, although adult stem (ADS) cells provide an alternative, more ethically acceptable, source. The annual global 100 million human birth rate proposes umbilical cord blood (UCB) as the largest untouched stem cell source, with advantages of naive immune status and relatively unshortened telomere length. Here, we report the worlds first reproducible production of cells expressing embryonic stem cell markers, – cord‐blood‐derived embryonic‐like stem cells (CBEs). UCB, after elective birth by Caesarean section, has been separated by sequential immunomagnetic removal of nucleate granulocytes, erythrocytes and haemopoietic myeloid/lymphoid progenitors. After 7 days of high density culture in microflasks, (105 cells/ml, IMDM, FCS 10%, thrombopoietin 10 ng/ml, flt3‐ligand 50 ng/ml, c‐kit ligand 20 ng/ml). CBE colonies formed adherent to the substrata; these were maintained for 6 weeks, then were subcultured and continued for a minimum 13 weeks. CBEs were positive for TRA‐1‐60, TRA‐1‐81, SSEA‐4, SSEA‐3 and Oct‐4, but not SSEA‐1, indicative of restriction in the human stem cell compartment. The CBEs were also microgravity–bioreactor cultured with hepatocyte growth medium (IMDM, FCS 10%, HGF 20 ng/ml, bFGF 10 ng/ml, EGF 10 ng/ml, c‐kit ligand 10 ng/ml). After 4 weeks the cells were found to express characteristic hepatic markers, cytokeratin‐18, α‐foetoprotein and albumin. Thus, such CBEs are a viable human alternative from embryonic stem cells for stem cell research, without ethical constraint and with potential for clinical applications.

Trends in Androgen Prescribing in the United States, 2001 to 2011

Although commercial sales of androgen replacement therapy (ART) have increased substantially in recent years,1,2 to our knowledge, no national population-based studies of this treatment have been reported. In view of the conflicting evidence on the risks and benefits of ART,3-7 understanding androgen prescribing patterns in the United States is important from both a clinical and a public health perspective. We used data from Clinformatics DataMart (CDM), one of the nations largest commercial health insurance populations, to examine androgen prescribing patterns in the United States over the past decade.

Amino acid supplementation increases lean body mass, basal muscle protein synthesis, and insulin-like growth factor-I expression in older women.

CONTEXT Inadequate dietary protein intake has been implicated in sarcopenia. OBJECTIVE AND DESIGN The objectives of this study were to determine whether: 1) chronic essential amino acid (EAA) supplementation improves postabsorptive muscle protein fractional synthesis rate (FSR), lean body mass (LBM), and one-repetition maximum muscle strength, and androgen receptor and IGF-I muscle protein expression; and 2) the acute anabolic response to EAA ingestion is preserved after a 3-month supplementation period. Using a randomized, double-blinded, placebo-controlled design, older women (68 +/- 2 yr) were assigned to receive either placebo (n = 7), or 15 g EAA/d [supplemented treatment group (SUP)] (n = 7) for 3 months. Metabolic outcomes were assessed in association with stable isotope studies conducted at 0 and 3 months. SETTING The study was performed at The University of Texas Medical Branch General Clinical Research Center. RESULTS Ingestion of 7.5 g EAA acutely stimulated FSR in both groups at 0 months (P < 0.05). Basal FSR at 3 months was increased in SUP only. The magnitude of the acute response to EAA was unaltered after 3 months in SUP. LBM increased in SUP only (P < 0.05). One-repetition maximum strength remained unchanged in both groups. Basal IGF-I protein expression increased in SUP after 3 months (P = 0.05), with no changes in androgen receptor or total and phosphorylated Akt, mammalian target of rapamycin, S6 kinase, and 4E-binding protein. CONCLUSIONS EAA improved LBM and basal muscle protein synthesis in older individuals. The acute anabolic response to EAA supplementation is maintained over time and can improve LBM, possibly offsetting the debilitating effects of sarcopenia.

Risk of Myocardial Infarction in Older Men Receiving Testosterone Therapy

Background: Testosterone therapy for older men has increased substantially over the past decade. Research on the effects of testosterone therapy on cardiovascular outcomes has yielded inconsistent results. Objective: To examine the risk of myocardial infarction (MI) in a population-based cohort of older men receiving intramuscular testosterone. Method: Using a 5% national sample of Medicare beneficiaries, we identified 6355 patients treated with at least 1 injection of testosterone between January 1, 1997, and December 31, 2005. We matched this cohort to 19 065 testosterone nonusers at a 1:3 ratio based on a composite MI prognostic score. Patients were followed until December 31, 2005, or until they lost coverage from Medicare, enrolled in a health maintenance organization, experienced a MI, or died. Result: In a Cox regression analysis adjusting for demographic and clinical characteristics, receipt of testosterone therapy was not associated with an increased risk of MI (hazard ratio [HR] = 0.84; 95% CI = 0.69-1.02). In this analysis, there was an interaction between receipt of testosterone and quartile of risk of MI (P = 0.023). For men in the highest quartile of the MI prognostic score, testosterone therapy was associated with a reduced risk of MI (HR = 0.69; 95% CI = 0.53-0.92), whereas there was no difference in risk for the first (HR = 1.20; 95% CI = 0.88-1.67), second (HR = 0.94; 95% CI = 0.69-1.30), and third quartiles (HR = 0.78; 95% CI = 0.59-1.01). Conclusion: Older men who were treated with intramuscular testosterone did not appear to have an increased risk of MI. For men with high MI risk, testosterone use was modestly protective against MI.

The Role of Androgens and Estrogens on Healthy Aging and Longevity

Aging is associated with a loss of sex hormone in both men (andropause) and women (menopause). In men, reductions in testosterone can trigger declines in muscle mass, bone mass, and in physical function. In women, the impact of the loss of sex hormones, such as estradiol, on bone is well elucidated, but evidence is limited on whether the loss of estradiol negatively affects muscle mass and physical function. However, deficiencies in multiple anabolic hormones have been shown to predict health status and longevity in older persons. Thus, consideration should be given as to whether targeted hormone replacement therapies may prove effective at treating clinical conditions, such as age-related sarcopenia, cancer cachexia, and/or acute or chronic illnesses. If initiated carefully in the appropriate clinical population, hormone replacement therapies in men and women may prevent and reverse muscle and bone loss and functional declines and perhaps promote healthy aging and longevity.

Effect of Growth Hormone Replacement Therapy on Cognition after Traumatic Brain Injury

Traumatic brain injury (TBI) is a major public health issue, and yet medical science has little to offer for the persistent symptoms that prevent many of these individuals from fully re-entering society. Post-traumatic hypopituitarism, and specifically growth hormone deficiency (GHD), has been found in a large percentage of individuals with chronic moderate to severe TBI. Presently, there are no published treatment studies of hormone replacement in this population. In this study, 83 subjects with chronic TBI were screened for hypopituitarism. Forty-two subjects were found to have either GHD or GH insufficiency (GHI), of which 23 agreed to be randomized to either a year of GH replacement or placebo. All subjects completed the study with no untoward side effects from treatment. A battery of neuropsychological tests and functional measures were administered before and after treatment. Improvement was seen on the following tests: Dominant Hand Finger Tapping Test, Wechsler Adult Intelligence Scale III-Information Processing Speed Index, California Verbal Learning Test II, and the Wisconsin Card Sorting Test (executive functioning). The findings of this pilot study provide preliminary evidence suggesting that some of the cognitive impairments observed in persons who are GHD/GHI after TBI may be partially reversible with appropriate GH replacement therapy.

An overview of the endocrinology of skeletal muscle

Endocrine regulation of the balance between skeletal muscle anabolism and catabolism has been investigated extensively. Factors determining whether hormones exert anabolic or catabolic influences are multifaceted and often unclear. Testosterone, growth hormone, insulin and insulin-like growth factor-I have complex anabolic effects, some of which have only recently been elucidated, and are important regulators of muscle remodeling, whereas glucocorticoids have direct catabolic effects and induce muscle protein loss. The effects of estrogen are poorly understood and warrant further study. We review recent literature and evaluate the hormones driving skeletal muscle anabolism and catabolism, which ultimately dictate the endocrinology and metabolism of skeletal muscle in humans. Understanding hormonal regulation of skeletal muscle remodeling might facilitate development of improved hormone-mediated therapies for muscle wasting conditions.

Troglitazone inhibits progesterone production in porcine granulosa cells

Troglitazone (a thiazolidinedione that improves insulin resistance) lowers elevated androgen concentrations in women with polycystic ovarian syndrome. In this study, we assessed the direct effects of troglitazone on steroidogenesis in porcine granulosa cells. Troglitazone inhibited progesterone production in a doseand time-dependent manner (earliest effects at 4 h, maximum at 24 h) without affecting cell viability. Progesterone production was also inhibited by troglitazone in the presence of 25-hydroxycholesterol, indicating that the drug does not affect intracellular cholesterol transport. Troglitazone also inhibited FSHand forskolin-stimulated progesterone secretion. The reduced progesterone production was accompanied by marked elevations of pregnenolone concentrations, suggesting inhibition of 3b-hydroxysteroid dehydrogenase (3b-HSD). The activity of 3b-HSD in troglitazone-treated granulosa cells was decreased by more than 60%, compared with controls after 24 h. Troglitazone did not affect aromatase activity in porcine granulosa cells. In summary, troglitazone has direct effects on porcine granulosa cell steroidogenesis. The drug specifically inhibits 3b-HSD activity, resulting in impaired progesterone production. The clinical relevance of this direct in vitro effect on steroidogenesis needs further investigation. (Endocrinology 139: 4962–4966, 1998) T represent a new class of drugs that have been demonstrated to significantly improve the treatment of type 2 diabetes mellitus (1, 2). Thiazolidinediones act primarily at adipose and muscle tissue, where they increase insulin sensitivity at the postreceptor level (3). This improvement in insulin resistance is the basis for their use in diabetes. Thiazolidinediones are high-affinity ligands for the peroxisome-proliferator activated receptor g (4). Thiazolidinediones bind to and activate peroxisome-proliferator activated receptor g that forms a heterodimer with retinoic acid receptor and binds to an orphan nuclear receptor-binding motif [direct repeat one (DR-1)] in gene promoters (5). By acting at the transcriptional level, thiazolidinediones selectively increase the expression of genes that regulate glucose homeostasis (6), and they decrease the expression of genes that oppose insulin action (7). These agents also stimulate adipocyte differentiation from preadipocyte fibroblasts (8) and counteract negative effects of some cytokines on glucose and lipid metabolism (9). Because thiazolidinediones improve peripheral insulin resistance and decrease hyperinsulinemia, they may also be used for the treatment of several disorders other than type 2 diabetes. One such disease is polycystic ovarian disease (PCO), where insulin resistance in peripheral tissues and resulting hyperinsulinemia are associated with increased androgen concentrations and oligomenorrhea (10). The thiazolidinedione troglitazone, when used for the treatment of PCO, improved insulin resistance and lowered androgen concentrations (11, 12). This beneficial effect of troglitazone was attributed to its lowering of serum insulin concentrations that are proposed to hyperstimulate the ovary (11, 12). Another possibility is that troglitazone affects ovarian function by direct effects on steroidogenesis. In this study, we used porcine granulosa cells in primary culture to investigate the in vitro effects of troglitazone on progesterone production. We found that troglitazone inhibits progesterone production in granulosa cells by inhibiting the activity of 3bhydroxysteroid dehydrogenase (3b-HSD). Materials and Methods

Directed engineering of umbilical cord blood stem cells to produce C-peptide and insulin

Abstract.  Objectives: In this study, we investigated the potential of umbilical cord blood stem cell lineages to produce C‐peptide and insulin. Materials and methods: Lineage negative, CD133+ and CD34+ cells were analyzed by flow cytometry to assess expression of cell division antigens. These lineages were expanded in culture and subjected to an established protocol to differentiate mouse embryonic stem cells (ESCs) toward the pancreatic phenotype. Phase contrast and fluorescence immunocytochemistry were used to characterize differentiation markers with particular emphasis on insulin and C‐peptide. Results: All 3 lineages expressed SSEA‐4, a marker previously reported to be restricted to the ESC compartment. Phase contrast microscopy showed all three lineages recapitulated the treatment‐dependent morphological changes of ESCs as well as the temporally restricted expression of nestin and vimentin during differentiation. After engineering, each isolate contained both C‐peptide and insulin, a result also obtained following a much shorter protocol for ESCs. Conclusions: Since C‐peptide can only be derived from de novo synthesis and processing of pre‐proinsulin mRNA and protein, we conclude that these results are the first demonstration that human umbilical cord blood‐derived stem cells can be engineered to engage in de novo synthesis of insulin.

A SELECTIVE SEROTONIN REUPTAKE INHIBITOR, FLUOXETINE HYDROCHLORIDE, MODULATES THE PULSATILE RELEASE OF PROLACTIN IN POSTMENOPAUSAL WOMEN

We investigated serotoninergic regulation of prolactin release in estrogen-withdrawn postmenopausal women by using a serotonin reuptake inhibitor. Subjects underwent frequent blood sampling after placebo (basal) or fluoxetine administration. Mean 24-hour serum prolactin concentrations increased significantly in response to fluoxetine. Objective pulse analysis revealed no change in prolactin pulse frequency with serotoninergic stimulation, but maximal serum prolactin peak heights increased significantly. Multiple-parameter deconvolution disclosed no change in prolactin half-life, but a significant increase in the total mass of prolactin secreted per 24 hours during fluoxetine administration. Cosinor analysis of the prolactin time series showed a significant increase in the circadian amplitude and mean without any change in the time of maximal concentration during treatment with fluoxetine. We conclude that short-term activation of the serotoninergic system in the absence of substantial estradiol and opiatergic tone significantly increases the secretion of prolactin in postmenopausal women.