JD induced pluripotent stem cell-derived hepatocytes faithfully recapitulate the pathophysiology of familial hypercholesterolemia.

UNLABELLED: Elevated levels of low-density lipoprotein cholesterol (LDL-C) in plasma are a major contributor to cardiovascular disease, which is the leading cause of death worldwide. Genome-wide association studies (GWAS) have identified 95 loci that associate with control of lipid/cholesterol metabolism. Although GWAS results are highly provocative, direct analyses of the contribution of specific allelic variations in regulating LDL-C has been challenging due to the difficulty in accessing appropriate cells from affected patients. The primary cell type responsible for controlling cholesterol and lipid flux is the hepatocyte. Recently, we have shown that cells with hepatocyte characteristics can be generated from human induced pluripotent stem cells (iPSCs). This finding raises the possibility of using patient-specific iPSC-derived hepatocytes to study the functional contribution of GWAS loci in regulating lipid metabolism. To test the validity of this approach, we produced iPSCs from JD a patient with mutations in the low-density lipoprotein receptor (LDLR) gene that result in familial hypercholesterolemia (FH). We demonstrate that (1) hepatocytes can be efficiently generated from FH iPSCs; (2) in contrast to control cells, FH iPSC-derived hepatocytes are deficient in LDL-C uptake; (3) control but not FH iPSC-derived hepatocytes increase LDL uptake in response to lovastatin; and (4) FH iPSC-derived hepatocytes display a marked elevation in secretion of lipidated apolipoprotein B-100. CONCLUSION: Cumulatively, these findings demonstrate that FH iPSC-derived hepatocytes recapitulate the complex pathophysiology of FH in culture. These results also establish that patient-specific iPSC-derived hepatocytes could be used to definitively determine the functional contribution of allelic variation in regulating lipid and cholesterol metabolism and could potentially provide a platform for the identification of novel treatments of cardiovascular disease. (HEPATOLOGY 2012).

Valproic acid activates Notch1 signaling and induces apoptosis in medullary thyroid cancer cells.

OBJECTIVE: To examine the effects of valproic acid (VPA) on Notch1 expression and cancer cell proliferation in medullary thyroid cancer (MTC) cells. BACKGROUND: Other than surgery, there are no effective treatments for MTC, a neuroendocrine malignancy that frequently metastasizes. We have previously shown that over-expression of Notch1 in MTC cells inhibits cell growth and hormone production. VPA, a drug long used for the treatment of epilepsy, has recently been identified as a potential Notch1 activator. We hypothesized that VPA might activate Notch1 signaling in MTC cells, with antiproliferative effects. METHODS: Human MTC cells were treated with VPA (0-5 mM) and Western blotting was performed to measure levels of Notch1 pathway proteins and neuroendocrine tumor markers. After confirming that VPA is a Notch1 activator in MTC cells, we performed cell proliferation assay. Finally, to determine the mechanism of growth inhibition, we measured protein levels of various markers of apoptosis. RESULTS: Notch1 was absent in MTC cells at baseline. VPA treatment resulted in an increase in both full-length and active Notch1 protein. Notch1 activation with VPA suppressed 2 neuroendocrine tumor markers, ASCL1 and chromogranin A. Importantly, VPA inhibited the growth of MTC cells in a dose-dependent manner. Immunoblot analysis demonstrated caspase activation and poly(ADP-ribose) polymerase cleavage, indicating the induction of apoptosis. CONCLUSIONS: VPA activates Notch1 signaling in MTC cells and inhibits their growth by inducing apoptosis. As the safety of VPA in human beings is well established, a clinical trial using this drug to treat patients with advanced MTC could be initiated in the near future.

A Drug Screen using Human iPSC-Derived Hepatocyte-like Cells Reveals Cardiac Glycosides as a Potential Treatment for Hypercholesterolemia.

Efforts to identify pharmaceuticals to treat heritable metabolic liver diseases have been hampered by the lack of models. However, cells with hepatocyte characteristics can be produced from induced pluripotent stem cells (iPSCs). Here, we have used hepatocyte-like cells generated from homozygous familial hypercholesterolemia (hoFH) iPSCs to identify drugs that can potentially be repurposed to lower serum LDL-C. We found that cardiac glycosides reduce the production of apolipoprotein B (apoB) from human hepatocytes in culture and the serum of avatar mice harboring humanized livers. The drugs act by increasing the turnover of apoB protein. Analyses of patient medical records revealed that the treatment of patients with cardiac glycosides reduced serum LDL-C levels. These studies highlight the effectiveness of using iPSCs to screen for potential treatments for inborn errors of hepatic metabolism and suggest that cardiac glycosides could provide an approach for reducing hepatocyte production of apoB and treating hypercholesterolemia.

Mapping the Cell-Surface N-Glycoproteome of Human Hepatocytes Reveals Markers for Selecting a Homogeneous Population of iPSC-Derived Hepatocytes.

When comparing hepatic phenotypes between iPSC-derived hepatocyte-like cells from different liver disease patients, cell heterogeneity can confound interpretation. We proposed that homogeneous cell populations could be generated by fluorescence-activated cell sorting (FACS). Using cell-surface capture proteomics, we identified a total of 300 glycoproteins on hepatocytes. Analyses of the expression profiles during the differentiation of iPSCs revealed that SLC10A1, CLRN3, and AADAC were highly enriched during the final stages of hepatocyte differentiation. FACS purification of hepatocyte-like cells expressing SLC10A1, CLRN3, or AADAC demonstrated enrichment of cells with hepatocyte characteristics. Moreover, transcriptome analyses revealed that cells expressing the liver gene regulatory network were enriched while cells expressing a pluripotent stem cell network were depleted. In conclusion, we report an extensive catalog of cell-surface N-linked glycoproteins expressed in primary hepatocytes and identify cell-surface proteins that facilitate the purification of homogeneous populations of iPSC-derived hepatocyte-like cells.

Aneuploidy is permissive for hepatocyte-like cell differentiation from human induced pluripotent stem cells.

BACKGROUND: The characterization of induced pluripotent stem cells (iPSCs) and embryonic stem cells (ESCs) routinely includes analyses of chromosomal integrity. The belief is that pluripotent stem cells best suited to the generation of differentiated derivatives should display a euploid karyotype; although, this does not appear to have been formally tested. While aneuploidy is commonly associated with cell transformation, several types of somatic cells, including hepatocytes, are frequently aneuploid and variation in chromosomal content does not contribute to a transformed phenotype. This insight has led to the proposal that dynamic changes in the chromosomal environment may be important to establish genetic diversity within the hepatocyte population and such diversity may facilitate an adaptive response by the liver to various insults. Such a positive contribution of aneuploidy to liver function raises the possibility that, in contrast to existing dogma, aneuploid iPSCs may be capable of generating hepatocyte-like cells that display hepatic activities. RESULTS: We examined whether a human iPSC line that had multiple chromosomal aberrations was competent to differentiate into hepatocytes and found that loss of normal chromosomal content had little impact on the production of hepatocyte-like cells from iPSCs. CONCLUSIONS: iPSCs that harbor an abnormal chromosomal content retain the capacity to generate hepatocyte-like cells with high efficiency.

Stable, dependable fixation of short-stem femoral implants at 5 years.

Conventional uncemented femoral implants provide dependable long-term fixation in patients with a wide range of clinical function. However, challenges with proximal-distal femoral mismatch, preservation of bone stock, and minimally invasive approaches have led to exploration into various other implant designs. Short-stem designs focusing on a stable metaphyseal fit have emerged to address these challenges in total hip arthroplasty (THA). The purpose of this study was to present the 5-year clinical and radiographic results of a computed tomography-based, custom-made, metaphyseal-engaging short-stem femoral implant.Sixty-one patients with an average age of 61 years (range, 22-75 years) and average body mass index of 28.9 kg/m(2) (range, 20.3-44.1 kg/m(2)) at follow-up underwent 69 THAs with the metaphyseal-engaging short stem. Clinical performance was evaluated using the Harris Hip Score and Western Ontario and McMaster Universities Arthritis Index score, and radiographs were reviewed for stability and bony ingrowth. Harris Hip Score averaged 55 (range, 20-90) preoperatively and 96 (range, 55-100) postoperatively. Western Ontario and McMaster Universities Arthritis Index score averaged 51 (range, 13-80) preoperatively and 3 (range, 0-35) postoperatively. No cases of subsidence were observed, and no revision surgeries were performed. Bone remodeling was typified by endosteal condensation and cortical hypertrophy in Gruen zones 2, 3, 5, and 6. At 5-year follow-up, the uncemented, metaphyseal-engaging short stem was stable and exhibited proximal bone remodeling closer to the metaphysis than conventional stems. Short-stem, metaphyseal-engaging femoral implants can meet the goals of a successful THA.

Thyroid hormone replacement in women of reproductive age: is surgeon knowledge related to operative volume?

BACKGROUND: It is not known if surgeons who place women of reproductive age on thyroid hormone replacement (levothyroxine, LT4) routinely inform them of the increased LT4 demands with future pregnancy. In addition, it is not clear whether reading the 2007 Endocrine Society's "Guidelines on the Management of Thyroid Dysfunction during Pregnancy and Postpartum" influences the role of the surgeon in patient education. The objective of this study was to identify the role of the surgeon in patient education at the time of initiation of LT4 therapy. METHODS: A two-sided one-page survey was distributed to the 260 surgeons attending the 2009 American Association of Endocrine Surgeons' meeting. Of those distributed, 109 (42%) surgeons returned the survey. RESULTS: Although only 26/109 (23%) read the 2007 Endocrine Society's "Guidelines on the Management of Thyroid Dysfunction during Pregnancy and Postpartum," reading the guidelines was associated with a significantly greater likelihood of informing patients of higher LT4 requirements with pregnancy (p < 0.0001). Eighty-five percent of those who read the guidelines "often" or "always" informed patients of higher LT4 requirements with future pregnancy, whereas only 44% of those who did not read the guidelines provided the same level of education. After controlling for surgeon sex, academic versus private practice, years in practice, and number of surgeries performed in 2008, the only independent predictor of patient education was reading the guidelines (p = 0.006). On multivariable analysis, surgeon volume was associated with reading the guidelines (p = 0.006). The mean number of thyroid surgeries performed by those who read the guidelines versus those who did not was 164.46 +/- 16.98 versus 115.01 +/- 8.27 (p = 0.005). CONCLUSION: Surgeons who read the Endocrine Society's "Guidelines on the Management of Thyroid Dysfunction during Pregnancy and Postpartum" were significantly more likely to inform patients of the need for higher LT4 doses with pregnancy. Thyroid surgery volume was associated with guideline awareness.

Sodium butyrate activates Notch1 signaling, reduces tumor markers, and induces cell cycle arrest and apoptosis in pheochromocytoma.

BACKGROUND: Pheochromocytoma is a neuroendocrine (NE) tumor of the adrenal medulla for which surgical resection is the only therapy. However, 10-46% of tumors are metastatic or have malignant features, and are often inoperable. Our lab has demonstrated the importance of the Notch1 signaling pathway in NE neoplasia, indicating that this pathway could be a target for emergent treatments in pheochromocytoma. It has recently become clear that histone deacetylase (HDAC) inhibitors influence Notch1 signaling. We hypothesized that the HDAC inhibitor Sodium Butyrate (NaB) might activate Notch1 in pheochromocytoma resulting in altered tumor cell proliferation. METHODS: Pheochromocytoma (PC-12) cells were treated with increasing concentrations of NaB. MTT cellular proliferation assay was used to determine the effect of NaB on PC-12 cell growth. Expression of Notch1, NE markers, and cell cycle proteins was studied using Western analysis. RESULTS: Untreated PC-12 cells lack Notch1 activity. Treatment with NaB led to a dose-dependent induction of Notch1 signaling, reduction of NE markers ASCL1 and CgA, and a significant reduction in cellular proliferation. Levels of expression of cyclin D1, p21, cleaved PARP, and cleaved caspase 3 proteins indicated the presence of cell cycle arrest and apoptosis following NaB treatment. CONCLUSION: NaB activated Notch1 signaling, inhibited cellular proliferation, reduced NE markers, and induced cell cycle arrest and apoptosis in pheochromocytoma cells. This data indicates that activation of Notch1 signaling is a promising potential therapy or palliative measure for pheochromocytoma that warrants further investigation.