Jaspal S. Khillan

ACCELERATED LIVER REGENERATION AND HEPATOCARCINOGENESIS IN MICE OVEREXPRESSING SERINE-45 MUTANT BETA-CATENIN

The Wnt/β‐catenin pathway is implicated in the pathogenesis of hepatocellular cancer (HCC). We developed a transgenic mouse (TG) in the FVB strain that overexpresses Ser45‐mutated‐β‐catenin in hepatocytes to study the effects on liver regeneration and cancer. In the two independent TG lines adult mice show elevated β‐catenin at hepatocyte membrane with no increase in the Wnt pathway targets cyclin‐D1 or glutamine synthetase. However, TG hepatocytes upon culture exhibit a 2‐fold increase in thymidine incorporation at day 5 (D5) when compared to hepatocytes from wildtype FVB mice (WT). When subjected to partial hepatectomy (PH), dramatic increases in the number of hepatocytes in S‐phase are evident in TG at 40 and WT at 72 hours. Coincident with the earlier onset of proliferation, we observed nuclear translocation of β‐catenin along with an increase in total and nuclear cyclin‐D1 protein at 40 hours in TG livers. To test if stimulation of β‐catenin induces regeneration, we used hydrodynamic delivery of Wnt‐1 naked DNA to control mice, which prompted an increase in Wnt‐1, β‐catenin, and known targets, glutamine synthetase (GS) and cyclin‐D1, along with a concomitant increase in cell proliferation. β‐Catenin‐overexpressing TG mice, when followed up to 12 months, showed no signs of spontaneous tumorigenesis. However, intraperitoneal delivery of diethylnitrosamine (DEN), a known carcinogen, induced HCC at 6 months in TG mice only. Tumors in TG livers showed up‐regulation of β‐catenin, cyclin‐D1, and unique genetic aberrations, whereas other canonical targets were unremarkable. Conclusion: β‐Catenin overexpression offers growth advantage during liver regeneration. Also, whereas no spontaneous HCC is evident, β‐catenin overexpression makes TG mice susceptible to DEN‐induced HCC. HEPATOLOGY 2010

A novel signaling by vitamin A/retinol promotes self renewal of mouse embryonic stem cells by activating PI3K/Akt signaling pathway via insulin-like growth factor-1 receptor.

Pluripotent embryonic stem (ES) cells are a potential source of all types of cells for regenerative medicine. ES cells maintain pluripotency through a complex interplay of different signaling pathways and transcription factors, including leukemia inhibitory factor (LIF), Nanog, Sox2, and Oct3/4. Nanog, however, plays a key role in maintaining the pluripotency of mouse and human ES cells. Phosphoinositde 3‐kinase (PI3K) signaling pathway which is activated in response to growth factors and cytokines also plays a critical role in promoting the survival and proliferation of ES cells. Our earlier studies revealed that retinol, the alcohol form of vitamin A, enhances the expression of Nanog and prevents differentiation of ES cells in long‐term cultures. Normally vitamin A/retinol is associated with cell differentiation via its potent metabolite, retinoic acid. Thus far, no direct function has been ascribed to retinol itself. In this study, we demonstrate for the first time that retinol directly activates phosphoinositide three (PI3) kinase signaling pathway through IGF‐1 receptor/insulin receptor substrate one (IRS‐1) by engaging Akt/PKB‐mTORC1 mammalian target of rapamycin‐2 (mammalian target of rapamycin complex 2), indicating a growth factor‐like function of vitamin A. Furthermore, ES cells do not express enzymes to metabolize retinol into retinoic acid and lack receptors for retinol transport into the cytoplasm, indicating that retinol signaling is independent of retinoic acid. This study presents a novel system to investigate how extracellular signals control the self renewal of ES cells which will be important for high‐quality ES cells for regenerative medicine. STEM CELLS 2010;28:57–63

Suppression of liver regeneration and hepatocyte proliferation in hepatocyte‐targeted glypican 3 transgenic mice

Glypican 3 (GPC3) belongs to a family of glycosylphosphatidylinositol‐anchored, cell‐surface heparan sulfate proteoglycans. GPC3 is overexpressed in hepatocellular carcinoma. Loss‐of‐function mutations of GPC3 result in Simpson‐Golabi‐Behmel syndrome, an X‐linked disorder characterized by overgrowth of multiple organs, including the liver. Our previous study showed that GPC3 plays a negative regulatory role in hepatocyte proliferation, and this effect may involve CD81, a cell membrane tetraspanin. To further investigate GPC3 in vivo, we engineered transgenic (TG) mice overexpressing GPC3 in the liver under the control of the albumin promoter. GPC3 TG mice with hepatocyte‐targeted, overexpressed GPC3 developed normally in comparison with their nontransgenic littermates but had a suppressed rate of hepatocyte proliferation and liver regeneration after partial hepatectomy. Moreover, gene array analysis revealed a series of changes in the gene expression profiles in TG mice (both in normal mice and during liver regeneration). In unoperated GPC3 TG mice, there was overexpression of runt related transcription factor 3 (7.6‐fold), CCAAT/enhancer binding protein alpha (2.5‐fold), GABA A receptor (2.9‐fold), and wingless‐related MMTV integration site 7B (2.8‐fold). There was down‐regulation of insulin‐like growth factor binding protein 1 (8.4‐fold), Rab2 (5.6‐fold), beta‐catenin (1.7‐fold), transforming growth factor beta type I (3.1‐fold), nodal (1.8‐fold), and yes‐associated protein (1.4‐fold). Changes after hepatectomy included decreased expression in several cell cycle–related genes. Conclusion: Our results indicate that in GPC3 TG mice, hepatocyte overexpression of GPC3 suppresses hepatocyte proliferation and liver regeneration and alters gene expression profiles, and potential cell cycle–related proteins and multiple other pathways are involved and affected. (HEPATOLOGY 2010;52:1060–1067)

Promotion of Feeder‐Independent Self‐Renewal of Embryonic Stem Cells by Retinol (Vitamin A)

Retinol, the alcohol form of vitamin A, maintains pluripotency of mouse embryonic stem cells (ESCs) by the overexpression of Nanog, which is a key transcription factor for their self‐renewal. ESCs represent the most promising source of all types of cells for regenerative medicine and drug discovery. These cells maintain pluripotency through a complex interplay of different signaling pathways and transcription factors including leukemia inhibitory factor (LIF), homeodomain protein Nanog, and Oct3/4. Nanog, however, plays a key role in maintaining the pluripotency of mouse and human ESCs. Overexpression of nanog by heterologous promoters can maintain pluripotency of ESCs in the absence of LIF. Also, Nanog alone is sufficient for the self‐renewal of ESCs while maintaining the Oct4 levels. Normally, mouse and human ESCs are cultured over mouse embryonic fibroblasts as feeders to maintain pluripotency. Although feeder cells provide important growth‐promoting factors, their use involves several cumbersome and time‐consuming steps. Here we demonstrate that retinol can support feeder‐independent self‐renewal of ESCs in long‐term cultures without affecting their pluripotency. The effect of retinol is independent of the strain background, and the cells maintain complete potential to differentiate into all the primary germ layers in embryoid bodies and in chimeric animals. Self‐renewal of ESCs by retinol is not mediated by retinoic acid. The studies demonstrate for the first time that a physiologically relevant small molecule has growth‐promoting effect on the self‐renewal of ESCs by activating the endogenous machinery to overexpress a critical gene for pluripotency.

Genes inducing iPS phenotype play a role in hepatocyte survival and proliferation in vitro and liver regeneration in vivo

Reprogramming factors have been used to induce pluripotent stem cells as an alternative to somatic cell nuclear transfer technology in studies targeting disease models and regenerative medicine. The neuronal repressor RE‐1 silencing transcription factor (REST) maintains self‐renewal and pluripotency in mouse embryonic stem cells by maintaining the expression of Oct3/4, Nanog, and cMyc. We report that primary hepatocytes express REST and most of the reprogramming factors in culture. Their expression is up‐regulated by hepatocyte growth factor (HGF) and epidermal growth factor (EGF). REST inhibition results in down‐regulation of reprogramming factor expression, increased apoptosis, decreased proliferation, and cell death. The reprogramming factors are also up‐regulated after 70% partial hepatectomy in vivo. Conclusion: These findings show that genes inducing the iPS phenotype, even though expressed at lower levels than embryonic stem cells, nonetheless are associated with control of apoptosis and cell proliferation in hepatocytes in culture and may play a role in such processes during liver regeneration. (HEPATOLOGY 2011)

Vitamin A/Retinol and Maintenance of Pluripotency of Stem Cells

Retinol, the alcohol form of vitamin A is a key dietary component that plays a critical role in vertebrate development, cell differentiation, reproduction, vision and immune system. Natural and synthetic analogs of retinol, called retinoids, have generally been associated with the cell differentiation via retinoic acid which is the most potent metabolite of retinol. However, a direct function of retinol has not been fully investigated. New evidence has now emerged that retinol supports the self-renewal of stem cells including embryonic stem cells (ESCs), germ line stem cells (GSCs) and cancer stem cells (CSCs) by activating the endogenous machinery for self-renewal by a retinoic acid independent mechanism. The studies have also revealed that stem cells do not contain enzymes that are responsible for metabolizing retinol into retinoic acid. This new function of retinol may have important implications for stem cell biology which can be exploited for quantitative production of pure population of pluripotent stem cells for regenerative medicine as well as clinical applications for cancer therapeutics.

Generation of chondrocytes from embryonic stem cells.

Pluripotent embryonic stem (ES) cells have complete potential for all the primary germ layers, such as ectoderm, mesoderm, and endoderm. However, the cellular and molecular mechanisms that control their lineage-restricted differentiation are not understood. Although embryoid bodies, which are formed because of the spontaneous differentiation of ES cells, have been used to study the differentiation into different cell types, including neurons, chondrocytes, insulin-producing cells, bone-forming cells, hematopoietic cells, and so on, this system has limitations for investigating the upstream events that lead to commitment of cells that occur during the inaccessible period of development. Recent developments in human ES cells have offered a challenge to develop strategies for understanding the basic mechanisms that play a key role in differentiation of stem cell into specific cell types for their applications in regenerative medicine and cell-based therapies. A micromass culture system was developed to induce the differentiation of ES cells into chondrocytes, the cartilage-producing cells, as a model to investigate the upstream events of stem cell differentiation. ES cells were co-cultured with limb bud progenitor cells. A high percentage of differentiated cells exhibit typical morphological characteristics of chondrocytes and express cartilage matrix genes such as collagen type II and proteoglycans, suggesting that signals from the progenitor cells are sufficient to induce ES cells into the chondrogenic lineage. Degeneration of cartilage in the joints is associated with osteoarthritis, which affects the quality of life of human patients. Therefore, the quantitative production of chondrocytes can be a powerful resource to alleviate the suffering of those patients.

Amplification of tumor inducing putative cancer stem cells (CSCs) by vitamin A/retinol from mammary tumors

Solid tumors contain a rare population of cancer stem cells (CSCs) that are responsible for relapse and metastasis. The existence of CSC however, remains highly controversial issue. Here we present the evidence for putative CSCs from mammary tumors amplified by vitamin A/retinol signaling. The cells exhibit mammary stem cell specific CD29(hi)/CD49f(hi)/CD24(hi) markers, resistance to radiation and chemo therapeutic agents and form highly metastatic tumors in NOD/SCID mice. The cells exhibit indefinite self renewal as cell lines. Furthermore, the cells exhibit impaired retinol metabolism and do not express enzymes that metabolize retinol into retinoic acid. Vitamin A/retinol also amplified putative CSCs from breast cancer cell lines that form highly aggressive tumors in NOD SCID mice. The studies suggest that high purity putative CSCs can be isolated from solid tumors to establish patient specific cell lines for personalized therapeutics for pre-clinical translational applications. Characterization of CSCs will allow understanding of basic cellular and molecular pathways that are deregulated, mechanisms of tumor metastasis and evasion of therapies that has direct clinical relevance.

Maintenance of feeder free anchorage independent cultures of ES and iPS cells by retinol/vitamin A

Pluripotent embryonic stem (ES) cells derived from mammalian blastocyst and the adult fibroblast derived induced pluripotent stem (iPS cells) exhibit complete potential to form cells representing all the primary germ layers such as mesoderm, endoderm and ectoderm. These cells are usually co‐cultured with mouse embryonic fibroblast feeders to prevent spontaneous differentiation. Feeder free cultures can provide substantial advantage to improve the efficiency and consistency of the culture conditions. In these studies, we demonstrate that a small dietary compound retinol, the alcohol form of vitamin A has capacity to regulate the pluripotency of pluripotent stem cells and maintain highly enriched population of pluripotent ES and iPS cells in feeder free suspension cultures. Retinol maintains long‐term cultures of undifferentiated cells via elevated expression of stem cell specific transcription factors Nanog and Oct4. The studies provide evidence that retinol regulates the self‐renewal of pluripotent stem cells via the over expression of insulin like growth factor II (IGFII) that engages PI3 kinase signaling pathway via IGF1 receptor tyrosine kinase. The ES cells retain capacity to generate high degree germline competent chimeric animals after microinjection into blastocysts. The studies offer a convenient system for long term maintenance of pluripotent stem cells via the activation of intracellular machinery for self‐renewal by a physiologically relevant compound for large‐scale production of high quality pluripotent stem cells. J. Cell. Biochem. 113: 3002–3010, 2012.

Differentiation of Embryonic Stem Cells into Cartilage Cells

Embryonic stem (ES) cells have complete potential to form all types of cells. Although these cells have indefinite capacity for self-renewal, the mechanisms that control their lineage-restricted differentiation are not well understood. Due to their potential to form all types of cells, these cells are expected to have applications in regenerative medicine to cure human diseases. Osteoarthritis (OA) is a degenerative disease of articular cartilage of weight bearing joints. Approximately twenty million people suffer from this debilitating disease. Therefore, the induced differentiation of ES cells into cartilage-producing cells will have potential application to cure OA. This unit describes a system to induce differentiation of a high percentage of ES cells into mesenchymal cells that differentiate into chondrocytes, the cartilage-producing cells. A quantitative production of chondrocytes can be a powerful resource to alleviate the suffering of those patients with OA. Furthermore, this can be an excellent system to investigate the upstream events of cell-restricted differentiation during the inaccessible period of development.