Jorge David Rivas-Carrillo

Reversal of mouse hepatic failure using an implanted liver-assist device containing ES cell–derived hepatocytes

Severe acute liver failure, even when transient, must be treated by transplantation and lifelong immune suppression. Treatment could be improved by bioartificial liver (BAL) support, but this approach is hindered by a shortage of human hepatocytes. To generate an alternative source of cells for BAL support, we differentiated mouse embryonic stem (ES) cells into hepatocytes by coculture with a combination of human liver nonparenchymal cell lines and fibroblast growth factor-2, human activin-A and hepatocyte growth factor. Functional hepatocytes were isolated using albumin promoter–based cell sorting. ES cell–derived hepatocytes expressed liver-specific genes, secreted albumin and metabolized ammonia, lidocaine and diazepam. Treatment of 90% hepatectomized mice with a subcutaneously implanted BAL seeded with ES cell–derived hepatocytes or primary hepatocytes improved liver function and prolonged survival, whereas treatment with a BAL seeded with control cells did not. After functioning in the BAL, ES cell–derived hepatocytes developed characteristics nearly identical to those of primary hepatocytes.

A human β-cell line for transplantation therapy to control type 1 diabetes

A human pancreatic β-cell line that is functionally equivalent to primary β-cells has not been available. We established a reversibly immortalized human β-cell clone (NAKT-15) by transfection of primary human β-cells with a retroviral vector containing simian virus 40 large T-antigen (SV40T) and human telomerase reverse transcriptase (hTERT) cDNAs flanked by paired loxP recombination targets, which allow deletion of SV40T and TERT by Cre recombinase. Reverted NAKT-15 cells expressed β-cell transcription factors (Isl-1, Pax 6, Nkx 6.1, Pdx-1), prohormone convertases 1/3 and 2, and secretory granule proteins, and secreted insulin in response to glucose, similar to normal human islets. Transplantation of NAKT-15 cells into streptozotocin-induced diabetic severe combined immunodeficiency mice resulted in perfect control of blood glucose within 2 weeks; mice remained normoglycemic for longer than 30 weeks. The establishment of this cell line is one step toward a potential cure of diabetes by transplantation.

Differentiation of mouse embryonic stem cells to hepatocyte-like cells by co-culture with human liver nonparenchymal cell lines

This protocol describes a co-culture system for the in vitro differentiation of mouse embryonic stem cells into hepatocyte-like cells. Differentiation involves four steps: (i) formation of embryoid bodies (EB), (ii) induction of definitive endoderm from 2-d-old EBs, (iii) induction of hepatic progenitor cells and (iv) maturation into hepatocyte-like cells. Differentiation is completed by 16 d of culture. EBs are formed, and cells can be induced to differentiate into definitive endoderm by culture in Activin A and fibroblast growth factor 2 (FGF-2). Hepatic differentiation and maturation of cells is accomplished by withdrawal of Activin A and FGF-2 and by exposure to liver nonparenchymal cell-derived growth factors, a deleted variant of hepatocyte growth factor (dHGF) and dexamethasone. Approximately 70% of differentiated embryonic stem (ES) cells express albumin and can be recovered by albumin promoter-based cell sorting. The sorted cells produce albumin in culture and metabolize ammonia, lidocaine and diazepam at approximately two-thirds the rate of primary mouse hepatocytes.

PuraMatrix facilitates bone regeneration in bone defects of calvaria in mice.

Artificial bones have often used for bone regeneration due to their strength, but they cannot provide an adequate environment for cell penetration and settlement. We therefore attempted to explore various materials that may allow the cells to penetrate and engraft in bone defects. PuraMatrix™ is a self-assembling peptide scaffold that produces a nanoscale environment allowing both cellular penetration and engraftment. The objective of this study was to investigate the effect of PuraMatrix™ on bone regeneration in a mouse bone defect model of the calvaria. Matrigel™ was used as a control. The expression of bone-related genes (alkaline phosphatase, Runx2, and Osterix) in the PuraMatrix™-injected bone defects was stronger than that in the Matrigel™-injected defects. Soft X-ray radiographs revealed that bony bridges were clearly observed in the defects treated with PuraMatrix™, but not in the Matrigel™-treated defects. Notably, PuraMatrix™ treatment induced mature bone tissue while showing cortical bone medullary cavities. The area of newly formed bones at the site of the bone defects was 1.38-fold larger for PuraMatrix™ than Matrigel™. The strength of the regenerated bone was 1.72-fold higher for PuraMatrix™ (146.0 g) than for Matrigel™ (84.7 g). The present study demonstrated that PuraMatrix™ injection favorably induced functional bone regeneration.Artificial bones have often used for bone regeneration due to their strength, but they cannot provide an adequate environment for cell penetration and settlement. We therefore attempted to explore various materials that may allow the cells to penetrate and engraft in bone defects. PuraMatrix™ is a self-assembling peptide scaffold that produces a nanoscale environment allowing both cellular penetration and engraftment. The objective of this study was to investigate the effect of PuraMatrix™ on bone regeneration in a mouse bone defect model of the calvaria. Matrigel™ was used as a control. The expression of bone-related genes (alkaline phosphatase, Runx2, and Osterix) in the PuraMatrix™-injected bone defects was stronger than that in the Matrigel™-injected defects. Soft X-ray radiographs revealed that bony bridges were clearly observed in the defects treated with PuraMatrix™, but not in the Matrigel™-treated defects. Notably, PuraMatrix™ treatment induced mature bone tissue while showing cortical bone medullary cavities. The area of newly formed bones at the site of the bone defects was 1.38-fold larger for PuraMatrix™ than Matrigel™. The strength of the regenerated bone was 1.72-fold higher for PuraMatrix™ (146.0 g) than for Matrigel™ (84.7 g). The present study demonstrated that PuraMatrix™ injection favorably induced functional bone regeneration.

Differentiation of human embryonic stem cells to hepatocytes using deleted variant of HGF and poly-amino-urethane-coated nonwoven polytetrafluoroethylene fabric.

Human embryonic stem (hES) cells have recently been studied as an attractive source for the development of a bioartificial liver (BAL). Here we evaluate the differentiation capacity of hES cells into hepatocytes. hES cells were subjected to suspension culture for 5 days, and then cultured onto poly-amino-urethane (PAU)-coated, nonwoven polytetrafluoroethylene (PTFE) fabric in the presence of fibroblast growth factor-2 (bFGF) (100 ng/ml) for 3 days, then with deleted variant of hepatocyte growth factor (dHGF) (100 ng/ml) and 1% dimethyl sulfoxide (DMSO) for 8 days, and finally with dexamethasone (10–7 M) for 3 days. The hES cells showed gene expression of albumin in a time-dependent manner of the hepatic differentiation process. The resultant hES-derived hepatocytes metabolized the loaded ammonia and lidocaine at 7.8% and 23.6%, respectively. A million of such hepatocytes produced albumin and urea at 351.2 ng and urea at 7.0 μg. Scanning electron microscopy showed good attachment of the cells on the surface of the PTFE fabric and well-developed glycogen rosettes and Gap junction. In the present work we have demonstrated the efficient differentiation of hES cells to functional hepatocytes. The findings are useful to develop a BAL.

Reestablishment of Microenvironment is Necessary to Maintain In Vitro and In Vivo Human Islet Function

Islet transplantation is associated with an elevated rate of early graft failure. The isolation process leads to structural and functional abnormalities. The reestablishment of the cell–matrix relationship is important to modulate the survival and function of islets. Thus, we evaluated the effect of human fibronectin (hFN) and self-assembling peptide nanofiber (SAPNF) in the ability to support islet function in vitro and after transplantation into streptozotocin (STZ)-induced diabetic severe combined immunodeficiency (SCID) mice. Human isolated islets were cultured with hFN or SAPNF for 7 days. Their ability to maintain insulin production/glucose responsiveness over time was evaluated. Islets embedded in hFN, SAPNF, or alone were transplanted into STZ-induced diabetic SCID mice. Islet grafts were removed after 14 days to evaluate insulin content, insulin expression, and apoptosis. SAPNF-entrapped islets maintained satisfactory morphology/viability and capability of glucose-dependent insulin secretion for over 7 days, whereas islets cultured in hFN underwent widespread deterioration. In vivo grafts containing human islets in SAPNF showed remarkably higher insulin content and expression when compared with human islets in hFn or alone. RT-PCR revealed lower caspase-3 expression in SAPNF islets grafts. These studies indicate that the reestablishment of the cell–matrix interactions by a synthetic matrix in the immediate postisolation period is a useful tool to maintain islet functions in vitro and in vivo.

Instant hepatic differentiation of human embryonic stem cells using activin A and a deleted variant of HGF

Human embryonic stem (hES) cells have the ability to differentiate into a variety of different cell lineages and potentially provide a source of differentiated cells for many therapeutic uses. Here we investigated an efficient method of hepatic differentiation from hES cells. A human ES cell line, KhES-1, was used and maintained by a nonfeeder method. KhES-1 cells were cultured for 5 days in the presence of human activin A (50 ng/ml) and then treated with a deleted variant of hepatocyte growth factor (dHGF) at 0, 100, or 500 ng/ml for 7 days. The resultant cells were biologically analyzed. The expression of the endodermal genes SOX17 and FOXA2 increased in KhES-1 cells after activin A treatment. In contrast, Oct4, a self-renewal undifferentiated marker, decreased in a time-dependent manner in KhES-1 cells. Following a 7-day treatment of the resultant cells with dHGF, especially at 500 ng/ml, KhES-1 cells showed an expression of the hepatic makers albumin, AFP, and CK18. Transitional electron microscopy showed well-developed glycogen rosettes and a gap junction in KhES-1 cells treated with 500 ng/ml of dHGF. We developed an efficient method to differentiate KhES-1 cells into hepatocyte-like cells in vitro using 50 ng/ml of activin A and 500 ng/ml of dHGF.

Self-Assembling Peptide Nanofiber as a Novel Culture System for Isolated Porcine Hepatocytes

Freshly isolated porcine hepatocytes are a very attractive cell source in the cell-based therapies to treat liver failure because of unlimited availability. However, due to the loss of hepatocyte functions in vitro, there is a need to develop a functional culture system to keep the cells metabolically active. Here we compared the effect of a self-assembling peptide nanofiber (SAPNF) as an extracellular matrix (ECM) with collagen type I on hepatocyte metabolic and secretion activities following hepatocyte isolation. Isolated porcine hepatocytes were cultured in SAPNF and collagen type I. Morphological assessment at different time points was performed by using SEM and phase contrast microscope. Metabolic and secretion activities were comparatively performed in the groups, by means of ammonia, lidocaine, and diazepam as well as albumin. Hepatocytes cultured on SAPNF revealed a three-dimensional spheroidal formation, thus maintaining cell differentiation status during 2 weeks of culture. On the other hand, hepatocytes in collagen revealed a spread shape, and by day 14 no hepatocyte-like cells were observed, but cells with long shape were present, thus revealing a degree of dedifferentiation in collagen culture. Hepatocytes in SAPNF were capable of drug-metabolizing activities and albumin secretion in higher ratio than those cultured on collagen. The present work clearly demonstrates the usefulness of SAPNF for maintaining differentiated functions of porcine hepatocytes in culture.

Transplantation of human hepatocytes cultured with deleted variant of hepatocyte growth factor prolongs the survival of mice with acute liver failure.

Background. Considering the scarcity of donor livers, it is extremely important to establish a functional culture method for isolated hepatocytes. As a tool for maintaining hepatocyte functions in vitro, dHGF, a variant of HGF (hepatocyte growth factor) with a deletion of five amino acids, attracted our attention because it is less cytotoxic compared with HGF. Methods. We evaluated growth, albumin production, metabolizing abilities of ammonia, lidocaine, and diazepam of human hepatocytes in the presence of dHGF (10–1000 ng/ml). The gene expression of liver markers was comparatively analyzed. The effect of intrasplenic transplantation of dHGF-treated human hepatocytes into severe combined immunodeficient (SCID) mice was evaluated in an acute liver failure (ALF) model induced by D-galactosamine (D-gal). Results. When 100 ng/ml of dHGF was utilized, metabolism rates of ammonia, lidocaine, and diazepam and albumin production per unit cell significantly increased. The gene expression analysis demonstrated the enhanced expression of albumin, HNF-4α, and C/EBPα in the hepatocytes treated with 100 ng/ml of dHGF. Transplantation of such hepatocytes prolonged the survival of the SCID mice with ALF induced by D-gal. Conclusions. The present work clearly demonstrates the usefulness of dHGF (100 ng/ml) for maintaining the differentiated functions of human hepatocytes in tissue culture.

Functional hepatocyte culture and its application to cell therapies.

Since Berry and Friend developed methods to isolate hepatocytes from the liver by a collagenase digestion technique in 1969, studies in laboratory animals have demonstrated that hepatocyte transplantation could potentially be used for the treatment of liver failure and inborn errors of liver-based metabolism. Healthy human hepatocytes are an ideal source for hepatocyte transplantation; however, their relative scarcity is one of the major drawbacks, further compounded by the competing demands of liver transplantation. Notably, most of the hepatocytes are isolated from discarded livers that are not suitable for organ transplantation for a variety of reasons, including excessive fat content. Importantly, the hepatocyte isolation procedure itself exerts major stress on hepatocytes by the disruption of cell-to-cell and cell-to-matrix contacts, resulting in hepatocytic apoptosis. Prevention of apoptosis would maximize yield of healthy cells and maintain hepatocyte differentiated function in culture. In this review, we describe methods to prevent apoptosis by utilizing both antiapoptotic molecules and matrices. We also introduce a new type of liver tissue engineering, hepatocyte sheet transplantation, which utilizes unwoven cloth having a cellular adhesive property.