Concise review: clinical programs of stem cell therapies for liver and pancreas.

Regenerative medicine is transitioning into clinical programs using stem/progenitor cell therapies for repair of damaged organs. We summarize those for liver and pancreas, organs that share endodermal stem cell populations, biliary tree stem cells (hBTSCs), located in peribiliary glands. They are precursors to hepatic stem/progenitors in canals of Hering and to committed progenitors in pancreatic duct glands. They give rise to maturational lineages along a radial axis within bile duct walls and a proximal-to-distal axis starting at the duodenum and ending with mature cells in the liver or pancreas. Clinical trials have been ongoing for years assessing effects of determined stem cells (fetal-liver-derived hepatic stem/progenitors) transplanted into the hepatic artery of patients with various liver diseases. Immunosuppression was not required. Control subjects, those given standard of care for a given condition, all died within a year or deteriorated in their liver functions. Subjects transplanted with 100-150 million hepatic stem/progenitor cells had improved liver functions and survival extending for several years. Full evaluations of safety and efficacy of transplants are still in progress. Determined stem cell therapies for diabetes using hBTSCs remain to be explored but are likely to occur following ongoing preclinical studies. In addition, mesenchymal stem cells (MSCs) and hematopoietic stem cells (HSCs) are being used for patients with chronic liver conditions or with diabetes. MSCs have demonstrated significant effects through paracrine signaling of trophic and immunomodulatory factors, and there is limited evidence for inefficient lineage restriction into mature parenchymal or islet cells. HSCs' effects are primarily via modulation of immune mechanisms.

Biliary tree stem cells, precursors to pancreatic committed progenitors: evidence for possible life-long pancreatic organogenesis.

Peribiliary glands (PBGs) in bile duct walls, and pancreatic duct glands (PDGs) associated with pancreatic ducts, in humans of all ages, contain a continuous, ramifying network of cells in overlapping maturational lineages. We show that proximal (PBGs)-to-distal (PDGs) maturational lineages start near the duodenum with cells expressing markers of pluripotency (NANOG, OCT4, and SOX2), proliferation (Ki67), self-replication (SALL4), and early hepato-pancreatic commitment (SOX9, SOX17, PDX1, and LGR5), transitioning to PDG cells with no expression of pluripotency or self-replication markers, maintenance of pancreatic genes (PDX1), and expression of markers of pancreatic endocrine maturation (NGN3, MUC6, and insulin). Radial-axis lineages start in PBGs near the ducts' fibromuscular layers with stem cells and end at the ducts' lumens with cells devoid of stem cell traits and positive for pancreatic endocrine genes. Biliary tree-derived cells behaved as stem cells in culture under expansion conditions, culture plastic and serum-free Kubota's Medium, proliferating for months as undifferentiated cells, whereas pancreas-derived cells underwent only approximately 8-10 divisions, then partially differentiated towards an islet fate. Biliary tree-derived cells proved precursors of pancreas' committed progenitors. Both could be driven by three-dimensional conditions, islet-derived matrix components and a serum-free, hormonally defined medium for an islet fate (HDM-P), to form spheroids with ultrastructural, electrophysiological and functional characteristics of neoislets, including glucose regulatability. Implantation of these neoislets into epididymal fat pads of immunocompromised mice, chemically rendered diabetic, resulted in secretion of human C-peptide, regulatable by glucose, and able to alleviate hyperglycemia in hosts. The biliary tree-derived stem cells and their connections to pancreatic committed progenitors constitute a biological framework for life-long pancreatic organogenesis.

Lineage restriction of human hepatic stem cells to mature fates is made efficient by tissue-specific biomatrix scaffolds.

UNLABELLED: Current protocols for differentiation of stem cells make use of multiple treatments of soluble signals and/or matrix factors and result typically in partial differentiation to mature cells with under- or overexpression of adult tissue-specific genes. We developed a strategy for rapid and efficient differentiation of stem cells using substrata of biomatrix scaffolds, tissue-specific extracts enriched in extracellular matrix, and associated growth factors and cytokines, in combination with a serum-free, hormonally defined medium (HDM) tailored for the adult cell type of interest. Biomatrix scaffolds were prepared by a novel, four-step perfusion decellularization protocol using conditions designed to keep all collagen types insoluble. The scaffolds maintained native histology, patent vasculatures, and ≈1% of the tissue's proteins but >95% of its collagens, most of the tissue's collagen-associated matrix components, and physiological levels of matrix-bound growth factors and cytokines. Collagens increased from almost undetectable levels to >15% of the scaffold's proteins with the remainder including laminins, fibronectins, elastin, nidogen/entactin, proteoglycans, and matrix-bound cytokines and growth factors in patterns that correlate with histology. Human hepatic stem cells (hHpSCs), seeded onto liver biomatrix scaffolds and in an HDM tailored for adult liver cells, lost stem cell markers and differentiated to mature, functional parenchymal cells in ≈1 week, remaining viable and with stable mature cell phenotypes for more than 8 weeks. CONCLUSION: Biomatrix scaffolds can be used for biological and pharmaceutical studies of lineage-restricted stem cells, for maintenance of mature cells, and, in the future, for implantable, vascularized engineered tissues or organs.

Multipotent stem/progenitor cells in human biliary tree give rise to hepatocytes, cholangiocytes, and pancreatic islets.

UNLABELLED: Multipotent stem/progenitors are present in peribiliary glands of extrahepatic biliary trees from humans of all ages and in high numbers in hepato-pancreatic common duct, cystic duct, and hilum. They express endodermal transcription factors (e.g., Sox9, SOX17, FOXA2, PDX1, HES1, NGN3, PROX1) intranuclearly, stem/progenitor surface markers (EpCAM, NCAM, CD133, CXCR4), and sometimes weakly adult liver, bile duct, and pancreatic genes (albumin, cystic fibrosis transmembrane conductance regulator [CFTR], and insulin). They clonogenically expand on plastic and in serum-free medium, tailored for endodermal progenitors, remaining phenotypically stable as undifferentiated cells for months with a cell division initially every ≈36 hours and slowing to one every 2-3 days. Transfer into distinct culture conditions, each comprised of a specific mix of hormones and matrix components, yields either cords of hepatocytes (express albumin, CYP3A4, and transferrin), branching ducts of cholangiocytes (expressing anion exchanger-2-AE2 and CFTR), or regulatable C-peptide secreting neoislet-like clusters (expressing glucagon, insulin) and accompanied by changes in gene expression correlating with the adult fate. Transplantation into quiescent livers of immunocompromised mice results in functional human hepatocytes and cholangiocytes, whereas if into fat pads of streptozocin-induced diabetic mice, results in functional islets secreting glucose-regulatable human C-peptide. CONCLUSION: The phenotypes and availability from all age donors suggest that these stem/progenitors have considerable potential for regenerative therapies of liver, bile duct, and pancreatic diseases including diabetes.

MAPK/ERK and Wnt/ß-Catenin pathways are synergistically involved in proliferation of Sca-1 positive hepatic progenitor cells.

Hepatic progenitor cells (HPCs) persist in adulthood and have the potential to play a major role in regenerating diseased liver. However, the signaling pathways that both directly and indirectly regulate HPCs' self-renewal and differentiation remain elusive. Previously, we identified a bipotent, stem cell antigen-1 (Sca-1) positive HPC population from naïve adult liver tissue. In the present study, we aimed to investigate the involvement of various signaling pathways in Sca-1(+) HPC proliferation. Epidermal growth factor (EGF) supplementation shows a significant increase in Sca-1(+) HPC proliferation and colony formation while stimulating phosphorylation of ERK1/2 and activating the induction of Cyclin D1. There were no demonstrable effects of EGF on Akt. The MEK inhibitor, PD0325901, inhibits proliferation and ERK1/2 phosphorylation while also suppressing the expression of Cyclin D1. In addition, activation of either IL-6/STAT3 or Wnt/ß-Catenin pathway did not independently support cell proliferation and colony formation of HPCs. The Wnt/ß-Catenin pathway can cooperate with EGF to significantly promote HPC colony formation ratio and maintain long-term HPC in vitro. The data indicates that the MAPK/ERK pathway is both essential and critical for HPC proliferation, and the Wnt signaling pathway is not sufficient, while it works synergistically with the MAPK/ERK signaling pathway to promote HPC proliferation.

Paracrine signals from mesenchymal cell populations govern the expansion and differentiation of human hepatic stem cells to adult liver fates.

UNLABELLED: The differentiation of embryonic or determined stem cell populations into adult liver fates under known conditions yields cells with some adult-specific genes but not others, aberrant regulation of one or more genes, and variations in the results from experiment to experiment. We tested the hypothesis that sets of signals produced by freshly isolated, lineage-dependent mesenchymal cell populations would yield greater efficiency and reproducibility in driving the differentiation of human hepatic stem cells (hHpSCs) into adult liver fates. The subpopulations of liver-derived mesenchymal cells, purified by immunoselection technologies, included (1) angioblasts, (2) mature endothelia, (3) hepatic stellate cell precursors, (4) mature stellate cells (pericytes), and (5) myofibroblasts. Freshly immunoselected cells of each of these subpopulations were established in primary cultures under wholly defined (serum-free) conditions that we developed for short-term cultures and were used as feeders with hHpSCs. Feeders of angioblasts yielded self-replication, stellate cell precursors caused lineage restriction to hepatoblasts, mature endothelia produced differentiation into hepatocytes, and mature stellate cells and/or myofibroblasts resulted in differentiation into cholangiocytes. Paracrine signals produced by the different feeders were identified by biochemical, immunohistochemical, and quantitative reverse-transcription polymerase chain reaction analyses, and then those signals were used to replace the feeders in monolayer and three-dimensional cultures to elicit the desired biological responses from hHpSCs. The defined paracrine signals were proved to be able to yield reproducible responses from hHpSCs and to permit differentiation into fully mature and functional parenchymal cells. CONCLUSION: Paracrine signals from defined mesenchymal cell populations are important for the regulation of stem cell populations into specific adult fates; this finding is important for basic and clinical research as well as industrial investigations.

NEMO-binding domain peptide promotes osteoblast differentiation impaired by tumor necrosis factor alpha.

Osteogenesis associated with persistent inflammation or infection exists in a broad range of conditions including rheumatoid arthritis and traumatic bone fracture. The poor outcomes of these conditions will benefit from more effective treatments. Here we investigated the molecular mechanisms and tested NEMO-binding domain peptide as a new approach of circumventing TNF-alpha inhibition of osteoblast differentiation. Our results showed: TNF-alpha markedly decreased BMP-2-induced alkaline phosphatase activity in the multipotent myoblast C2C12 cells in a dose dependent manner; stepwise experiments demonstrated that BMP-2-induced Smad1 activity was abrogated by addition of exogenous TNF-alpha or overexpression of NF-kappaB, and it was significantly elevated by overexpression of IkappaBalpha, an inhibitor of NF-kappaB; Western blotting showed that TNF-alpha markedly decreased the amount of phospho-Smad1 in BMP-2-activated C2C12 cells, but it did not alter Smad1 mRNA abundance as measured by real-time PCR; addition of a functional cell-permeable NEMO-binding domain (NBD) peptide antagonized NF-kappaB activity and ameliorated TNF-alpha inhibition of osteoblast differentiation. Taken together, our study reveals for the first time that NF-kappaB activation inhibits osteoblast differentiation by attenuating Smad1 activity and application of NBD peptide ameliorates this inhibitory effect. This could lead to new therapeutic drugs that circumvent the inflammatory inhibition of osteogenesis for treatment of traumatic open fractures with infection, rheumatoid arthritis and other bone loss disorders.

Structural insights into fibronectin type III domain-mediated signaling.

The alternatively spliced type III extradomain B (EIIIB) of fibronectin (FN) is expressed only during embryogenesis, wound healing and tumorigenesis. The biological function of this domain is unclear. We describe here the first crystal structure of the interface between alternatively spliced EIIIB and its adjacent FN type III domain 8 (FN B-8). The opened CC' loop of EIIIB, and the rotation and tilt of EIIIB allow good access to the FG loop of FN-8, which is normally hindered by the CC' loop of FN-7. In addition, the AGEGIP sequence of the CC'' loop of EIIIB replaces the NGQQGN sequence of the CC' loop of FN-7. Finally, the CC'' loop of EIIIB forms an acidic groove with FN-8. These structural findings warrant future studies directed at identifying potential binding partners for FN B-8 interface, linking EIIIB to skeletal and cartilaginous development, wound healing, and tumorigenesis, respectively.

Transcriptional coactivation of bone-specific transcription factor Cbfa1 by TAZ.

Core-binding factor 1 (Cbfa1; also called Runx2) is a transcription factor belonging to the Runt family of transcription factors that binds to an osteoblast-specific cis-acting element (OSE2) activating the expression of osteocalcin, an osteoblast-specific gene. Using the yeast two-hybrid system, we identified a transcriptional coactivator, TAZ (transcriptional coactivator with PDZ-binding motif), that binds to Cbfa1. A functional relationship between Cbfa1 and TAZ is demonstrated by the coimmunoprecipitation of TAZ by Cbfa1 and by the fact that TAZ induces a dose-dependent increase in the activity of osteocalcin promoter-luciferase constructs by Cbfa1. A dominant-negative construct of TAZ in which the coactivation domains have been deleted reduces osteocalcin gene expression down to basal levels. NIH 3T3, MC 3T3, and ROS 17/2.8 cells showed the expected nuclear localization of Cbfa1, whereas TAZ was distributed throughout the cytoplasm with some nuclear localization when transfected with either Cbfa1 or TAZ. Upon cotransfection by both Cbfa1 and TAZ, the transfected TAZ shows predominant nuclear localization. The dominant-negative construct of TAZ shows minimal nuclear localization upon cotransfection with Cbfa1. These data indicate that TAZ is a transcription coactivator for Cbfa1 and may be involved in the regulation of osteoblast differentiation.

Donor-associated malignancy in kidney transplant patients.

Skin cancer cells with donor genotype have been identified in allogeneic transplant patients; however, the donor contribution to the recipient's epithelial malignancy remains to be established. In this issue of the JCI, Verneuil et al. provide the first evidence for donor contribution to the malignant epithelium of skin squamous cell carcinoma in a kidney transplant recipient. This case report may have important implications for cancer research and clinical care of long-surviving kidney transplant patients.