History and development of an integrated model of care for female pelvic medicine and surgery.

Urologists and gynecologists manage most of the patients with female pelvic floor disorders. However, lack of a single focused approach among these two disciplines and other related fields may hinder advances in clinical care and research. Herein, along with describing the background of birth of the new subspeciality, we present a practical approach by which we established an integrated model of care and discovery for FPFD at our institution. With the recent approval of this subspeciality by the Accreditation Council for Graduate Medical Education (ACGME), it is plausible that other institutions would wish to initiate similar steps toward establishment of an integrated model of care for FPFD, and hence move this new subspecialty to its new frontiers.

Transcriptional profiling and hepatogenic potential of acute hepatic failure-derived bone marrow mesenchymal stem cells.

UNLABELLED: Liver stem cell (LSC) transplantation is a promising alternate approach to liver transplantation for patients with end-stage liver disease. However, the precise origin of LSCs remains unclear. Herein we determine if bone marrow mesenchymal stem cells (BMSCs) isolated from rats in acute hepatic failure (AHF) possess hepatic characteristics and have differentiation potential. BMSCs were isolated from AHF and sham-operated rats, and primary hepatocytes were isolated from normal rats for comparison. The transcriptomic profile of BMSCs and primary hepatocytes was analyzed using the Affymetrix GeneChip Rat Genome 230 2.0 Array. BMSCs isolated from AHF and normal rats were induced to differentiate into hepatocytes in vitro and the degree of hepatic differentiation was assessed using quantitative real time RT-PCR, immunohistochemistry, and biochemical assays. AHF-derived BMSCs had a significantly different gene expression profile compared to control BMSCs. Thirty-four gene/probe sets were expressed in both AHF-derived BMSCs and primary hepatocytes, but were absent in control-derived BMSCs, including 3 hepatocyte-specific genes. Forty-four genes were up-regulated more than 2-fold in AHF-derived BMSCs compared to controls, including 3 genes involved in hepatocyte metabolism and development. Furthermore, AHF-derived BMSCs expressed more hepatocyte related genes than control BMSCs. Additional experiments to validate the differentiation of AHF-derived BMSCs, compared to control-derived BMSCs, showed that several hepatocyte-specific genes and proteins [such as albumin (ALB) and alpha fetoprotein (AFP)] were expressed earlier, and at higher levels, after 1 week of differentiation. Hepatocyte-specific metabolic functions were also significantly higher in the AHF group compared to control cells. CONCLUSION: AHF-derived BMSCs had a hepatic transcriptional profile and expressed hepatocyte specific genes early during differentiation, and possessed greater hepatogenic potency in vitro compared to cells isolated from control animals, further confirming their potential as a stem cell-based therapy for end-stage liver disease.

3D PLGA scaffolds improve differentiation and function of bone marrow mesenchymal stem cell-derived hepatocytes.

UNLABELLED: Liver tissue engineering with hepatic stem cells provides a promising alternative to liver transplantation in patients with acute and chronic hepatic failure. In this study, a three-dimensional (3D) bioscaffold was introduced for differentiation of rat bone marrow mesenchymal stem cells (BMSCs) into hepatocytes. For hepatocyte differentiation, third passage BMSCs isolated from normal adult F344 rats were seeded into collagen-coated poly(lactic-co-glycolic acid) (C-PLGA) 3D scaffolds with hepatocyte differentiation medium for 3 weeks. Hepatogenesis in scaffolds was characterized by reverse transcript PCR, western blot, confocal laser scanning microscopy (CLSM), periodic acid-Schiff staining, histochemistry, and biochemical assays with hepatic-specific genes and markers. A monolayer culture system was used as a control differentiation group. The results showed that isolated cells possessed the basic features of BMSCs. Differentiated hepatocyte-like cells in C-PLGA scaffolds expressed hepatocyte-specific markers [eg, albumin (ALB), alpha-fetoprotein, cytokeratin 18, hepatocyte nuclear factor 4alpha, and cytochrome P450] at mRNA and protein levels. Most markers were expressed in C-PLGA group 1 week earlier than in the control group. Results of biocompatibility indicated that the differentiated hepatocyte-like cells grew more stably in C-PLGA scaffolds than that in controls during a 3-week differentiation period. The significantly higher metabolic functions in hepatocyte-like cells in the C-PLGA scaffold group further demonstrated the important role of the scaffold. CONCLUSION: As the phenomenon of transdifferentiation is uncommon, our successful transdifferentiation rates of BMSCs to mature hepatocytes prove the superiority of the C-PLGA scaffold in providing a suitable environment for such a differentiation. This material can possibly be used as a bioscaffold for liver tissue engineering in future clinical therapeutic applications.

Factors released from cholestatic rat livers possibly involved in inducing bone marrow hepatic stem cell priming.

Previous studies have shown that bone marrow beta 2m(-)/Thy-1+ hepatic stem cells (BMHSCs) were able to engraft in vivo and differentiate into functioning hepatocytes in vitro. Our transcriptomic profiling on BMHSCs derived from rats subjected to common bile duct ligation (CBDL) demonstrated CBDL-derived beta 2m(-)/Thy-1+ BMHSCs expressed hepatocyte-like genes and shared more commonly expressed genes with hepatocytes, suggesting that an "on-site" priming of BMHSCs into hepatocyte lineage was initiated under the condition of CBDL. In this paper, transcriptomic profiling was carried out on livers from rats with CBDL to identify candidate factors released from cholestatic livers possibly involved in the priming of BMHSCs using Affymetrix Rat Genome U34A arrays. In CBDL rat livers, 1,091 probe sets were differentially expressed, of which 188 up-regulated probe sets were annotated as "extracellular" components. Gene ontology analysis showed many up-regulated genes belonged to cytokines, chemokines and growth factors, including Il1b, Il18, Ptn, Spp1, Grn, Ccl2, Cxcl1, Pf4, Tgfb, and Tgfb3. Cell differentiation and proliferation regulation factors such as Dmbt1, Efna1, Lgals1, Lep, Pmp2, and Gas6 were also induced in CBDL livers. Furthermore, many proteolysis and peptidolysis genes such as Mmp2, Mmp12, Mmp14, and Mmp23 were up-regulated in CBDL livers. Gene expression profiling showed that many cytokine-, chemokine-, growth factor- as well as certain extracellular protein-related genes were induced in CBDL livers, suggesting that these genes may be involved in hepatic BMHSCs priming.

Intrasplenic transplantation of encapsulated genetically engineered mouse insulinoma cells reverses streptozotocin-induced diabetes in rats.

Pancreatic islet transplantation is limited by shortage of donor organs. Although beta-cell lines could be used, their secretion of insulin is characteristically glucose independent and immunoisolation is required. Here we show that intrasplenic transplantation of encapsulated glucose-responsive mouse insulinoma cells reversed streptozotocin (STZ)-induced diabetes in rats. MIN-6 cells derived from a transgenic mouse expressing SV 40 large T antigen in pancreatic beta-cells were transfected with minigene encoding for human glucagon-like-peptide-1 under the control of rat insulin promoter. The cells were encapsulated in alginate/poly-L-lysine and used for cell transplantation in STZ-diabetic rats. Rats with nonfasting blood glucose (n-FBG) greater than 350 mg/dl were used. In group I rats (n=6) 20 million encapsulated cells were injected into the spleen. Group II rats (n=6) received empty capsules. n-FBG was measured biweekly. After 4 and 8 weeks, an intraperitoneal glucose tolerance test (IPGTT) was performed in group I; normal rats served as controls. Plasma insulin level was measured every other week (RIA). After 8 weeks, spleens were removed 1 day before sacrifice. In rats transplanted with cells the n-FBG was 100-150 mg/dl until the end of the study. After splenectomy, all cell recipients became diabetic (glucose 400 +/- 20 mg/dl). Transplanted rats showed increase in body weight and insulin production (3.3 +/- 1.0 ng/ml versus 0.92 +/- 0.3 ng/ml; p < 0.01) and had normal IPGTT. Spleens contained capsules with insulin-positive cells. Overall, data from this work indicate that intrasplenic transplantation of xenogeneic encapsulated insulin-producing cells without immunosuppression reversed diabetes in rats. Excellent survival and function of the transplanted cells was due to the fact that the cells were separated from the bloodstream by the immunoisolatory membrane only and insulin was delivered directly to the liver (i.e., in a physiological manner).

A novel molecular marker of pituitary tumor transforming gene involves in a rat liver regeneration.

BACKGROUND: Pituitary tumor transforming gene (PTTG), homologous to a mammalian securin, plays a pivotal role in cell transformation, however, its biological function(s) in normal tissues is not fully understood. Because the liver is a regenerative organ, the relevant biological function of PTTG in the liver would be more feasible to understand PTTG. Also, PTTG may be involved in the liver regeneration. MATERIALS AND METHODS: Expressions of rat hepatic PTTG messengerRNA (mRNA) and cellular immunoreactivities during the cell proliferative period of the liver regeneration both in vitro and in vivo were tested. RESULTS: PTTG expression of the rat primary hepatocyte was stimulated by HGF in a dose dependent manner, and was suppressed when hepatocyte proliferation was inhibited by transforming growth factor-beta1. A positive PTTG immunoreactive co-localizing with 5-bromo-2'-deoxyuridine (BrdU) in the hepatocyte nucleus was found and there was a concurrent sister chromatin itself by the immunofluorescent labeling of PTTG with cytokeratin 18 (CK18). DISCUSSION: Since the correlation of PTTG mRNA expression, cell proliferation and immunoreactivity were observed in primary rat cultured hepatocytes, PTTG may be a novel marker of cell proliferation both in vitro and in vivo liver regeneration.

Transcriptomic fingerprinting of bone marrow-derived hepatic beta2m-/Thy-1+ stem cells.

The aim of the present study was to determine if the bone marrow (BM) beta2m-/Thy-1+ stem cells isolated from common bile duct ligated (CBDL) rats possess hepatocyte-like characteristics in their global gene expression profiles. The Affymetrix RG U34A arrays were used to conduct transcriptomic profiling on BM beta2m-/Thy-1+ stem cells isolated from CBDL and control rats as well as primary hepatocytes. Forty-one probe sets were up-regulated more than 2-fold in CBDL-derived beta2m-/Thy-1+ BM stem cells compared to control BM stem cells. Twenty-seven probe sets were present in both CBDL-derived beta2m-/Thy-1+ BM stem cells and control hepatocytes but absent in control beta2m-/Thy-1+ BM stem cells, including Tcf1 and Dbp. Compared to the control beta2m-/Thy-1+ BM stem cells, CBDL-derived beta2m-/Thy-1+ BM stem cells shared more commonly expressed genes with hepatocytes. Overall, CBDL-derived beta2m-/Thy-1+ stem cells displayed a different transcriptomic fingerprint compared with beta2m-/Thy-1+ BM stem cells isolated from control rats; and CBDL-derived beta2m-/Thy-1+ stem cells started to express some hepatocyte-like genes.

A novel sub-population of bone marrow-derived myocardial stem cells: potential autologous cell therapy in myocardial infarction.

BACKGROUND: Several studies have identified beta2-microglobulin-negative (beta2M(-)) cells as a potential stem cell fraction in the bone marrow of rats and humans. We studied the ability of bone marrow-derived beta2M(-) cells to differentiate into cardiomyocytes and reconstitute the myocardium in a model of myocardial infarction. METHODS: beta2M(-) cells were purified from bone marrow of Lewis rats using a magnetic activated cell-sorting technique. beta2M(-) cells, 2.5 x 10(6) cells in 100 microl of phosphate-buffered saline (PBS), were transplanted 7 days after infarction into a transmural myocardial scar induced by cryoinjury in Lewis rats (n = 9). Control Group 1(n = 10) received a 100-microl injection of PBS, and Control Group 2 (n = 15) received no injection. The beta2M(-) cells were labeled before transplantation, using the membrane fluorescent intercalated dye, PKH26. Repopulation was examined at 6 and 8 weeks after transplantation. Differentiation of beta2M(-) cells into cardiac myocytes was determined by the colocalization of troponin and PKH26 to the same cell, utilizing immunohistochemistry, ultraviolet photomicroscopy and fluorescence microscopy on 6-microm serial sections. Area of engraftment within the scar was calculated by planimetry. RESULTS: The treatment group had multiple islands of de novo-formed myocardium within the fibrous matrix of the transmural scar (mean area 35 +/- 4.2% of scar area at 6 and 8 weeks). These cells colocalized cardiac-specific troponin and PKH26. Using these techniques, no myocardial islands were seen in the control groups. Before transplantation, beta2M(-) cells were troponin-negative. CONCLUSIONS: This study demonstrates that beta2M(-) cells represent a novel sub-population of bone marrow-derived stem cells capable of successful and substantial engraftment in areas of transmural myocardial scar, with de novo formation of cardiac myocytes. The functional significance of this observation is being studied.

Prospective, randomized, multicenter, controlled trial of a bioartificial liver in treating acute liver failure.

OBJECTIVE: The HepatAssist liver support system is an extracorporeal porcine hepatocyte-based bioartificial liver (BAL). The safety and efficacy of the BAL were evaluated in a prospective, randomized, controlled, multicenter trial in patients with severe acute liver failure. SUMMARY BACKGROUND DATA: In experimental animals with acute liver failure, we demonstrated beneficial effects of the BAL. Similarly, Phase I trials of the BAL in acute liver failure patients yielded promising results. METHODS: A total of 171 patients (86 control and 85 BAL) were enrolled. Patients with fulminant/subfulminant hepatic failure and primary nonfunction following liver transplantation were included. Data were analyzed with and without accounting for the following confounding factors: liver transplantation, time to transplant, disease etiology, disease severity, and treatment site. RESULTS: For the entire patient population, survival at 30 days was 71% for BAL versus 62% for control (P = 0.26). After exclusion of primary nonfunction patients, survival was 73% for BAL versus 59% for control (n = 147; P = 0.12). When survival was analyzed accounting for confounding factors, in the entire patient population, there was no difference between the 2 groups (risk ratio = 0.67; P = 0.13). However, survival in fulminant/subfulminant hepatic failure patients was significantly higher in the BAL compared with the control group (risk ratio = 0.56; P = 0.048). CONCLUSIONS: This is the first prospective, randomized, controlled trial of an extracorporeal liver support system, demonstrating safety and improved survival in patients with fulminant/subfulminant hepatic failure.

Intrasplenic transplantation of encapsulated cells: a novel approach to cell therapy.

Cell therapy is likely to succeed clinically if cells survive at the transplantation site and are protected against immune rejection. We hypothesized that this could be achieved with intrasplenic transplantation of encapsulated cells because the cells would have instant access to oxygen and nutrients while being separated from the host immune system. In order to provide proof of the concept, primary rat hepatocytes and human hepatoblastoma-derived HepG2 cells were used as model cells. Rat hepatocytes were encapsulated in 100-kDa hollow fibers and cultured for up to 28 days. Rat spleens were implanted with hollow fibers that were either empty or contained I x 10(7) rat hepatocytes. Human HepG2 cells were encapsulated using alginate/ poly-L-lysine (ALP) and also transplanted into the spleen; control rats were transplanted with free HepG2 cells. Blood human albumin levels were measured using Western blotting and spleen sections were immunostained for albumin. Hepatocytes in monolayer cultures remained viable for only 6-10 days, whereas the cells cultured in hollow fibers remained viable and produced albumin throughout the study period. Allogeneic hepatocytes transplanted in hollow fibers remained viable for 4 weeks (end of study). Free HepG2 transplants lost viability and function after 7 days, whereas encapsulated HepG2 cells remained viable and secreted human albumin at all time points studied. ALP capsules, with or without xenogeneic HepG2 cells, produced no local fibrotic response. These data indicate that intrasplenic transplantation of encapsulated cells results in excellent survival and function of the transplanted cells and that the proposed technique has the potential to allow transplantation of allo- and xenogeneic cells (e.g., pancreatic islets) without immunosuppression.

Glycerol suppresses proliferation of rat hepatocytes and human HepG2 cells.

BACKGROUND: In fulminant hepatic failure (FHF), the ability of surviving hepatocytes to proliferate is diminished. Therefore, it is important that medical therapy cause no further impairment of liver regeneration. In FHF, intracranial hypertension secondary to brain edema is the most common cause of brain injury and death and glycerol is used in some countries to treat this complication. Glycerol has been long known to suppress the growth of various cell types. We therefore decided to examine the effect of glycerol on hepatocyte proliferation in vitro and in vivo in rats subjected to partial (2/3) hepatectomy. Additionally, we investigated the effect of glycerol on the proliferation of HepG2 cells. MATERIALS AND METHODS: Mitogen-induced primary rat hepatocytes were cultured in a hormonally defined Dulbecco's modified Eagle's medium containing increasing amounts of glycerol (0.5, 1.0, 2.0, 4.0%). HepG2 cells were cultured in minimal essential medium/10% FBS. After 2 days, HepG2 cells were exposed to glycerol (1.0-2.0-4.0%) and harvested after 48 h. Control dishes contained no glycerol. Cell proliferation was measured by the incorporation of [(3)H]thymidine and/or bromodeoxyuridine (BrdU). In vivo, Sprague-Dawley rats were subjected to standard partial 2/3 hepatectomy and assigned to intraportal administration of either 400 microl of glycerol or saline. Rats were killed after 1, 2, 3, 5, and 7 days. Liver weight/body weight ratio and BrdU uptake were measured. RESULTS: In all cultures tested, glycerol suppressed the growth of cells in a dose-dependent manner. In vivo, a single intraportal dose of glycerol slowed the liver regenerative response. CONCLUSIONS: This study demonstrated that glycerol has a potent growth-inhibitory effect on hepatocyte proliferation in vivo and in vitro. Remarkably, glycerol inhibited the proliferation of liver cancer cells as well. The results of this study have important clinical implications.

Immediate early genes and p21 regulation in liver of rats with acute hepatic failure.

It has been observed that liver regeneration in acute hepatic failure (AHF) is suppressed [Eguchi et al. Hepatology 1996;24(6):1452-9]. The molecular mechanism regulating this inhibition is not known. We previously reported that in AHF rats, hepatocyte proliferation was significantly impaired with elevation in serum IL-6, TGF-beta1, and HGF [Kamohara et al. Biochem Biophys Res Commun 2000;273(1):129-35]. Following either 70% partial hepatectomy (PH) or liver injury, quiescent mature hepatocytes are "primed" to re-enter the cell cycle. The process of "priming" appears to be triggered by extracellular cytokines (IL-6 and TNF-alpha) and is characterized by expression of immediate early genes. Under the stimulation of growth factors such as HGF, "primed" hepatocytes exit the G1 phase of the cell cycle. G1-associated cyclins and their inhibitors play a pivotal role in G1/S cell cycle transition. Here, we demonstrate that immediate early gene (i.e. c-myc, c-fos) expression and AP-1 activity are preserved in AHF rat livers despite absence of hepatocyte proliferation. In contrast, p21 mRNA and protein are both over-expressed in AHF livers compared to livers from rats undergoing PH; this elevation leads to inhibition in Cdk2 activity, resulting in G1 cell cycle arrest and inhibition of regeneration.