Selecting serum-free hepatocyte cryopreservation stage and storage temperature for the application of an "off-the-shelf" bioartificial liver system.

The bioartificial liver (BAL) system can potentially rescue acute liver failure (ALF) patients by providing partial liver function until a suitable donor liver can be found or the native liver has self-regenerated. In this study, we established a suitable cryopreservation process for the development of an off-the-shelf BAL system. The viability of hepatocyte spheroids cryopreserved in liquid nitrogen was comparable to that of fresh primary hepatocyte spheroids. When hepatocyte spheroids were subjected to cryopreservation in a deep freezer, no statistically significant differences were observed in ammonia removal rate or urea secretion rate based on the cryopreservation period. However, the functional activity of the liver post-cryopreservation in a deep freezer was significantly lower than that observed following liquid nitrogen cryopreservation. Moreover, cryopreserving spheroid hydrogel beads in a deep freezer resulted in a significant decrease (approximately 30%) in both ammonia removal and urea secretion rates compared to the group cryopreserved in liquid nitrogen. The viabilities of spheroid hydrogel beads filled into the bioreactor of a BAL system were similar across all four groups. However, upon operating the BAL system for 24 h, the liver function activity was significantly higher in the group comprising hydrogel beads generated after thawing hepatocyte spheroids cryopreserved in liquid nitrogen. Consequently, the manufacturing of beads after the cryopreservation of hepatocyte spheroids is deemed the most suitable method, considering efficiency, economic feasibility, and liver function activity, for producing a BAL system.

Establishment of a Serum-Free Hepatocyte Cryopreservation Process for the Development of an "Off-the-Shelf" Bioartificial Liver System.

To use hepatocytes immediately when necessary for hepatocyte transplantation and bioartificial liver (BAL) systems, a serum-free cryopreservation protocol ensuring the high survival of hepatocytes and maintenance of their functions should be developed. We established a serum-free protocol for the cryopreservation of primary hepatocytes, hepatocyte spheroids, and hepatocyte spheroid beads in liquid nitrogen. The serum-free cryopreservation solutions showed a significantly higher performance in maintaining enhanced viability and ammonia removal, urea secretion, and the albumin synthesis of hepatocyte spheroids and spheroid beads. The serum-free thawing medium, containing human serum albumin (HSA) and N-acetylcysteine (NAC), was compared with a fetal bovine serum-containing thawing medium for the development of a serum-free thawing medium. Our results show that hepatocyte spheroids and spheroid beads thawed using a serum-free thawing medium containing HSA and NAC exhibited increased hepatocyte viability, ammonia removal, urea secretion, and albumin synthesis compared to those thawed using the serum-containing medium. Finally, we evaluated the liver functions of the cryopreserved BAL system-applied serum-free cryopreservation process compared to the fresh BAL system. The ammonia removal efficiency of the cryopreserved hepatocyte spheroids BAL was lower than or similar to that of the fresh BAL system. Additionally, the urea concentrations in the media of all three BAL systems were not significantly different during BAL system operation. This cryopreserved spheroid-based BAL system using a serum-free process will be a good candidate for the treatment of patients.

B7­H1­mediated immunosuppressive properties in human mesenchymal stem cells are mediated by STAT­1 and not PI3K/Akt signaling.

Mesenchymal stem cells (MSCs), derived from either bone marrow (BM) or Wharton's jelly (WJ), inhibit the proliferation of activated T cells, and interferon (IFN)­Î³ serves an important role in this process. This process is B7­homolog (H)1­dependent during cell contact inhibition. However, the signaling pathway involved in B7­H1 expression in MSCs remains largely undefined. The present study demonstrated activation of B7­H1 by engaging signal transducer and activator of transcription (STAT)­1 signaling in MSCs. Human BM­ and WJ­MSCs were isolated and cultured. The immunosuppressive effect of BM­ and WJ­MSCs on phytohemagglutinin (PHA)­induced T cell proliferation was compared using direct and indirect co­culture systems. B7­H1 expression on BM­ and WJ­MSCs was detected by flow cytometry. Small interfering (si)RNA was used to knock down the expression of STAT­1. The inhibitory effect of MSCs on T lymphocytes was observed using PHA­induced T cell proliferation assays. IFN­Î³­induced B7­H1 expression on human BM­ and WJ­MSCs increased in a time­dependent manner. Furthermore, the inhibitory effect of MSCs on T cell proliferation was be restored when an anti­B7­H1 monoclonal antibody was used. When STAT­1 signaling was inhibited by siRNA, B7­H1 expression on IFN­Î³­treated MSCs decreased and T cell proliferation was restored; however, the expression of B7­H1 did not alter upon treatment with a phosphatidylinositol­3­kinase (PI3K) inhibitor (LY294002). These results demonstrated that the IFN­Î³­induced immunosuppressive properties of B7­H1 in human BM­ and WJ­MSCs were mediated by STAT­1 signaling, and not by PI3K/RAC­α serine/threonine­protein kinase signaling. Understanding the intracellular mechanisms underlying IFN­Î³­induced expression of B7­H1 in MSCs may ultimately lead to an improved understanding of MSCs and provide insight into their use as cell therapy agents.

Enhanced Immunosuppressive Properties of Human Mesenchymal Stem Cells Primed by Interferon-γ.

Mesenchymal stem cells (MSCs) are of particular interest for the treatment of immune-related diseases owing to their immunosuppressive properties. In this study, we aimed to identify the effect of interferon (IFN)-γ priming on immunomodulation by MSCs and elucidate the possible mechanism underlying their properties for the clinical treatment of allogeneic conflicts. Infusion of MSCs primed with IFN-γ significantly reduced the symptoms of graft-versus-host disease (GVHD) in NOD-SCID mice, thereby increasing survival rate when compared with naïve MSC-infused mice. However, infusion of IFN-γ-primed MSCs in which indoleamine 2,3-dioxygenase (IDO) was downregulated did not elicit this effect. The IDO gene was expressed in MSCs via the IFN-γ-Janus kinase (JAK)-signal transducer and activator of transcription 1 (STAT1) pathway, and the infusion of IDO-over-expressing MSCs increased survival rate in an in vivo GVHD model, similar to infusion of IFN-γ-primed MSCs. These data indicate that IFN-γ production by activated T-cells is correlated with the induction of IDO expression in MSCs via the IFN-γ-JAK-STAT1 pathway, which in turn results in the suppression of T-cell proliferation. Our findings also suggest that cell therapy based on MSCs primed with IFN-γ can be used for the clinical treatment of allogeneic conflicts, including GVHD.

Functional Evaluation of a Bioartificial Liver Support System Using Immobilized Hepatocyte Spheroids in a Porcine Model of Acute Liver Failure.

Bioartificial livers (BAL) may offer acute liver failure (ALF) patients an opportunity for cure without liver transplantation. We evaluated the efficacy of a spheroid-based BAL system, containing aggregates of porcine hepatocytes, in a porcine model of ALF. ALF pigs were divided into three groups. The control group consisted of treatment naïve pigs (n = 5), blank group consisted of pigs that were attached to the BAL system not containing hepatocytes for 12 hours (n = 5) and BAL group consisted of pigs that were attached to the BAL containing hepatocytes for 12 hours (n = 5). Increase in serum ammonia levels were significantly greater in the blank group (P < 0.01) and control group (P < 0.01), compared to the BAL group during the treatment period. Increase in ICP was significantly greater in the control group compared to the BAL group (P = 0.01). Survival was significantly prolonged in the BAL group compared to the blank group (P = 0.03). A BAL system with a bioreactor containing hepatocyte spheroids showed effective clearance of serum ammonia, preservation of renal function and delayed ICP increase in a porcine model of ALF.

Effect of ex vivo culture conditions on immunosuppression by human mesenchymal stem cells.

A microarray analysis was performed to investigate whether ex vivo culture conditions affect the characteristics of MSCs. Gene expression profiles were mainly influenced by the level of cell confluence rather than initial seeding density. The analysis showed that 276 genes were upregulated and 230 genes downregulated in MSCs harvested at ~90% versus ~50% confluence (P < 0.05, FC > 2). The genes that were highly expressed in MSCs largely corresponded to chemotaxis, inflammation, and immune responses, indicating direct or indirect involvement in immunomodulatory functions. Specifically, PTGES and ULBP1 were up-regulated in MSCs harvested at high density. Treatment of MSCs with PTGES or ULBP1 siRNA reversed their inhibition of T-cell proliferation in vitro. The culture conditions such as cell confluence at harvest seem to be important for gene expression profile of MSCs; therefore, the results of this study may provide useful guidelines for the harvest of MSCs that can appropriately suppress the immune response.

Rapid isolation of adipose tissue-derived stem cells by the storage of lipoaspirates.

PURPOSE: This study examined a rapid isolation method decreasing the time and cost of the clinical application of adipose tissue-derived stem cells (ASCs). MATERIALS AND METHODS: Aliquots (10 g) of the lipoaspirates were stored at 4°C without supplying oxygen or nutrients. At the indicated time points, the yield of mononuclear cells was evaluated and the stem cell population was counted by colony forming unit-fibroblast assays. Cell surface markers, stem cell-related transcription factors, and differentiation potentials of ASCs were analyzed. RESULTS: When the lipoaspirates were stored at 4°C, the total yield of mononuclear cells decreased, but the stem cell population was enriched. These ASCs expressed CD44, CD73, CD90, CD105, and HLA-ABC but not CD14, CD31, CD34, CD45, CD117, CD133, and HLA-DR. The number of ASCs increased 1×10(14) fold for 120 days. ASCs differentiated into osteoblasts, adipocytes, muscle cells, or neuronal cells. CONCLUSION: ASCs isolated from lipoaspirates and stored for 24 hours at 4°C have similar properties to ASCs isolated from fresh lipoaspirates. Our results suggest that ASCs can be isolated with high frequency by optimal storage at 4°C for 24 hours, and those ASCs are highly proliferative and multipotent, similar to ASCs isolated from fresh lipoaspirates. These ASCs can be useful for clinical application because they are time- and cost-efficient, and these cells maintain their stemness for a long time, like ASCs isolated from fresh lipoaspirates.

Effective myotube formation in human adipose tissue-derived stem cells expressing dystrophin and myosin heavy chain by cellular fusion with mouse C2C12 myoblasts.

Stem cell therapy for muscular dystrophies requires stem cells that are able to participate in the formation of new muscle fibers. However, the differentiation steps that are the most critical for this process are not clear. We investigated the myogenic phases of human adipose tissue-derived stem cells (hASCs) step by step and the capability of myotube formation according to the differentiation phase by cellular fusion with mouse myoblast C2C12 cells. In hASCs treated with 5-azacytidine and fibroblast growth factor-2 (FGF-2) for 1 day, the early differentiation step to express MyoD and myogenin was induced by FGF-2 treatment for 6 days. Dystrophin and myosin heavy chain (MyHC) expression was induced by hASC conditioned medium in the late differentiation step. Myotubes were observed only in hASCs undergoing the late differentiation step by cellular fusion with C2C12 cells. In contrast, hASCs that were normal or in the early stage were not involved in myotube formation. Our results indicate that stem cells expressing dystrophin and MyHC are more suitable for myotube formation by co-culture with myoblasts than normal or early differentiated stem cells expressing MyoD and myogenin.

Early Immunomodulation by Intravenously Transplanted Mesenchymal Stem Cells Promotes Functional Recovery in Spinal Cord Injured Rats.

Although intravenous administration of mesenchymal stem cells (MSCs) can enhance functional recovery after spinal cord injury (SCI), the underlying mechanisms have to be elucidated. In this study, we explored the mechanisms for functional recovery in SCI rats after intravenous transplantation of MSCs derived from human umbilical cord blood. Sprague-Dawley rats were randomly assigned to receive either MSCs (1 × 10(6) cells/0.5 ml) or PBS into the tail vein immediately after SCI. They were then evaluated by the Basso-Beattie-Bresnahan (BBB) locomotor rating scale weekly for 8 weeks and by somatosensory evoked potentials (SSEPs) 8 weeks after transplantation. MSC-treated rats showed a modest but significant improvement in BBB scores and latencies of SSEPs, compared with PBS controls. When human-specific Alu element was measured in the spinal cord, it was detected only 1 h after transplantation, suggesting transient engraftment of MSCs. Inflammatory cytokines were also determined using RT-PCR or Western blot in spinal cord extracts. In MSC-treated rats, the level of proinflammatory cytokine IL-1ß was decreased, but that of anti-inflammatory cytokine IL-10 was increased. MSCs also immediately suppressed IL-6 at 1 h posttransplantation. However, the response of IL-6, which has an immunoregulatory role, was increased 1-3 days after transplantation. In addition, we quantified microglia/macrophage stained with Iba-1 around the damaged spinal cord using immunohistochemistry. A proportion of activated microglia and macrophages in total Iba-1(+) cells was significantly decreased in MSC-treated rats, compared with PBS controls. These results suggest that early immunomodulation by intravenously transplanted MSCs is a potential underlying mechanism for functional recovery after SCI.

Hepatocyte transplantation for glycogen storage disease type Ib.

Glycogen storage disease type I (GSD-I) is a group of autosomal recessive disorders with an incidence of 1 in 100,000. The two major subtypes are GSD-Ia, caused by a deficiency of glucose-6-phosphatase (G6Pase), and GSD-Ib, caused by a deficiency of glucose-6-phosphate transporter (G6PT). We report that a substantial improvement was achieved following several infusions of hepatocytes in a patient with GSD-Ib. Hepatocytes were isolated from the unused cadaveric whole livers of two donors. At the first transplantation, approximately 2 x 10(9) cells (2% of the estimated recipient's total hepatocytes) were infused. Seven days later 1 x 10(9) (1% of liver mass) cryopreserved hepatocytes from the same donor were infused, and an additional 3 x 10(9) (3% of liver mass) cells from the second donor were infused 1 month after the second transplantation. After the hepatocyte transplantation, the patient showed no hypoglycemic symptoms despite the discontinuation of cornstarch meals. Liver biopsies on posttransplantation days 20 and 250 showed a normal level of glucose-6-phosphatase activity in presolubilization assay that was very low before transplantation. This was the first and successful clinical hepatocyte transplantation in Korea. In this study, hepatocyte transplantation allowed a normal diet in a patient with GSD-Ib, with substantial improvement in their quality of life. Hepatocyte transplantation might be an alternative to liver transplantation and dietary therapy in GSD-Ib.

Reconstruction of rabbit corneal epithelium on lyophilized amniotic membrane using the tilting dynamic culture method.

Rabbit corneal epithelium was reconstructed using tilting dynamic culture with a self-manufactured, amniotic membrane (AM) supporter and a lyophilized amniotic membrane (LAM). Rabbit corneal epithelial (RCE) cells were cultured and cryopreserved after isolation from the limbus. The second- and third-passage RCE cells were plated onto the epithelial side of the LAM of Ahn's AM supporter. Two days later, the air-liquid interface culture was maintained with third-passage RCE cells for 6 days and second-passage corneal epithelial cells for 9 days. The average viability of thawed RCE cells, assessed using trypan blue dye exclusion, was 77.42%. The reconstructed corneal epithelium was characterized by histological (hematoxylin and eosin) and immunohistochemical staining (proliferating cell nuclear antigen) for light microscopy, and by reverse transcriptase-polymerase chain reaction, glucose assay, and transmission electron microscopy. The basal layer of the reconstructed corneal epithelium was well formed, and the epithelium was tightly constructed due to the increase in cell proliferation and differentiation caused by the tilting dynamic culture, as opposed to static culture. Tilting dynamic culture was useful for the reconstruction of the epithelium using easily damaged epithelial cells and resulted in more stratum cell layers. Moreover, cytokeratin (CK3) mRNA expression in tilting dynamic cultured third-passage RCE cells seeded onto AM was greater than in static cultured third-passage RCE cells. The morphology of the reconstructed corneal epithelium on LAM by tilting dynamic culture for 9 days resembled that of the skin epidermis. This was thought to be because the tilting dynamic culture not only accelerated the proliferation and differentiation of cells by physical or mechanical stimulation, but also ensured that the supply of medium was delivered to the basal cells more efficiently. Thus, the reconstruction of the corneal epithelium using LAM and tilting dynamic culture was considered to be a good in vitro model for autologous or allogeneic transplantation of corneal epithelium and skin epidermis in patients with damaged epithelia.

Bioartificial liver systems: current status and future perspective.

Because the liver is a multifunctional and a vital organ for survival, the management of acute liver failure requires the support of a huge number of metabolic functions performed by the organ. Many early detoxification-based artificial liver techniques failed to treat the patients owing to the inadequate support of the many essential hepatic functions. For this reason, a bioartificial liver (BAL) comprising of viable hepatocytes on a mechanical support is believed to more likely provide these essential functions than a purely mechanical device. From 1990, nine clinical studies of various BAL systems have been reported, most of which utilize a hollow fiber technology, and a much larger number of various BAL systems have been suggested to show an enhanced performance. Safety issues such as immunological reactions, zoonosis and tumorgenicity have been successfully addressed for regulatory approval, but a recent report from a large-scale, randomized, and controlled phase III trial of a leading BAL system (HepatAssist) failed to meet our expectation of efficacy in terms of the overall survival rate. In this paper, we review the current BAL systems actively studied and discuss critical issues such as the hepatocyte bioreactor configuration and the hepatocyte source. On the basis of the insights gained from previously developed BAL systems and the rapid progress in stem cell technology, the short-term and long-term future perspectives of BAL systems are suggested.