Zun Chang Liu

Transdifferentiation of bioencapsulated bone marrow cells into hepatocyte-like cells in the 90% hepatectomized rat model

Under specific conditions, bone marrow cells can transdifferentiate into a variety of cell types including hepatocytes. In this study, bioencapsulated bone marrow cells were transplanted intraperitoneally into 90% hepatectomized rats. We then followed the transdifferentiation of the bone marrow cells and the effect of this on liver regeneration in this liver failure model. Bone marrow cells isolated from Wistar rats were bioencapsulated using alginate‐polylysine‐alginate method. These bioencapsulated bone marrow cells were transplanted intraperitoneally into 90% hepatectomized Wistar rats. Blood chemistry, HGF, liver weight, and survival of the recipient rats were evaluated. Histology and immunocytochemistry were used to analyze the bioencapsulated cells before and 14 days after transplantation. Unlike free bone marrow cells, transplantation of bioencapsulated bone marrow cells improved the survival of 90% hepatectomized rats and improved the blood chemistry with an efficacy similar to that of bioencapsulated hepatocytes or free hepatocytes transplantation. Some bioencapsulated bone marrow cells expressed hepatocytes markers of cytokeratins 8, cytokeratins 18, albumin, and AFP after 2 weeks of transplantation. These results suggest that syngeneic bioencapsulated bone marrow cells can transdifferentiate into hepatocyte‐like cells in the peritoneal cavity of 90% hepatectomized rats and increased the survival rates of these rats. In conclusion, these findings suggest the potential for a new alternative to hepatoctye transplantation for cellular therapy of acute liver failure. Liver Transpl 12:566–572, 2006.

Increased viability of transplanted hepatocytes when hepatocytes are co-encapsulated with bone marrow stem cells using a novel method.

This study is to investigate the viability of hepatocytes when transplanted into Wistar rats using co-encapsulated hepatocytes and bone marrow stem cells. Hepatocytes and bone marrow stem cells, isolated from Wistar rats, are co-encapsulated using either the standard single-step method or a novel two-step cell encapsulation method (www.artcell.mcgill.ca). After intraperitoneal transplantation into Wistar rats, the histology, fate of recovered microcapsules and viability of encapsulated hepatocytes are studied. When prepared using the standard method, there is excellent viability but only for up to 3 weeks. After this, there is extensive fibrous coating and severe fibrous adhesion and no microcapsules can be recovered. On the other hand, using the new two-step encapsulation method, the viability of the encapsulated hepatocytes can be followed for more than 4 months after transplantation. Even up to 4 months, there is significantly less host reaction when using the two-step encapsulation method and 50% of the microcapsules can be recovered. Co-encapsulated with bone marrow stem cells resulted in further increase in viability of the hepatocytes when followed up to 4 months after transplantation This new approach may improve the potential feasibility of using co-encapsulation of hepatocytes and bone marrow stem cells in bio-artificial liver support for the treatment of liver failure, especially for acute liver failure.

Evaluation of two protocols of uremic rat model: partial nephrectomy and infarction.

Animal models of chronic renal failure have been mostly achieved by partial ablation of renal parenchyma, the two most common techniques employed being surgical resection or infarction. Evaluation of the uremic model using these two techniques was carried out in Wistar rats. Two weeks after operative procedure, measured serum urea levels in the resection and infarction models were 59.1 and 64.3 mg/dL (normal range 15.6–24.4 mg/dL) respectively. However, the standard deviation in the former was significantly lower, 6.3 vs. 97.1 mg/dL from infarction model, p = 0.007. A consistent degree of glomerular filtration rate reduction was obtained in the resection model, resulting in 20–30% of normal creatinine clearance. This compared favorably with the creatinine clearance range (0.3–74% of normal) from the infarction model, in which two animals died of uremia and seven had higher than 50% of normal creatinine clearance. It is reasonable to attribute reproducibility and homogeneity demonstrated in the resection model to (i) more precise control of renal ablation extent with surgical techniques and (ii) less interplay of confounding injury mechanism to remnant kidney. These data support superiority of the resection model as an experimental tool for pathophysiological and/or interventional investigations of chronic renal failure.

Free and Microencapsulated Lactobacillus and Effects of Metabolic Induction on Urea Removal

We have previously reported the experimental use of genetically engineered Escherichia coli with microencapsulation to lower nitrogenous waste. Concern has surfaced, nonetheless, about safety of genetically engineered product. The purpose of this study is to explore the alternative use of probiotics in removal of plasma urea. After repeated cycles of exposure of Lactobacillus delbrueckii in urea‐rich medium under anaerobic environment, the organisms were demonstrated to lower plasma urea concentration in vitro. Suspension of Lactobacillus in uremic plasma reduced the urea nitrogen levels from 51.5 ± 5.2 mg/dL to 44.3 ± 3.9 mg/dL (P = 0.02) after 24 hours. With microencapsulation of Lactobacillus (inside semipermeable alginate‐polylysine‐alginate polymeric membrane), further lowering of urea nitrogen levels was achieved (35.4 ± 0.8 mg/dL, P = 0.03) at 24 hours. These preliminary data show that expression of certain enzymes could be induced in Lactobacillus delbrueckii and thus capable of lowering plasma urea. Further studies and molecular analysis would be indicated to explore and refine the techniques.

Long-term Effects on the Histology and Function of Livers and Spleens in Rats after 33% Toploading of PEG-PLA-nano Artificial Red Blood Cells

This study is to investigate the long-term effects of nanodimension PEG-PLA artificial red blood cells containing hemoglobin and red blood cell enzymes on the liver and spleen after 1/3 blood volume top loading in rats. The experimental rats received one of the following infusions: Nano artificial red blood cells in Ringer lactate, Ringer lactate, stroma-free hemoglobin, polyhemoglobin, and autologous rat whole blood. Blood samples were taken before infusions and on days 1, 7, and 21 after infusions for analysis. Nano artificial red blood cells, polyhemoglobin, Ringer lactate and rat red blood cells did not have any significant adverse effects on alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, creatine kinase, amylase and creatine kinase. On the other hand, stroma-free hemoglobin induced significant adverse effects on liver as shown by elevation in alanine aminotransferase and aspartate aminotransferase throughout the 21 days. On day 21 after infusions rats were sacrificed and livers and spleens were excised for histological examination. Nano artificial red blood cells, polyhemoglobin, Ringer lactate and rat red blood cells did not cause any abnormalities in the microscopic histology of the livers and spleens. In the stroma-free hemoglobin group the livers showed accumulation of hemoglobin in central veins and sinusoids, and hepatic steatosis. In conclusion, injected nano artificial red blood cells can be efficiently metabolized and removed by the reticuloendothelial system, and do not have any biochemical or histological adverse effects on the livers or the spleens.

Artificial Cell Microencapsulated Stem Cells in Regenerative Medicine, Tissue Engineering and Cell Therapy

Adult stem cells, especially isolated from bone marrow, have been extensively investigated in recent years. Studies focus on their multiple plasticity oftransdifferentiating into various cell lineages and on their potential in cellular therapy in regenerative medicine. In many cases, there is the need for tissue engineering manipulation. Among the different approaches of stem cells tissue engineering, microencapsulation can immobilize stem cells to provide a favorable microenvironment for stem cells survival and functioning. Furthermore, microencapsulated stem cells are immunoisolated after transplantation. We show that one intraperitoneal injection of microencapsulated bone marrow stem cells can prolong the survival of liver failure rat models with 90% of the liver removed surgically. In addition to transdifferentiation, bone marrow stem cells can act as feeder cells. For example, when coencapsulated with hepatocytes, stem cells can increase the viability and function of the hepatocytes in vitro and in vivo.

Preliminary Study on Intrasplenic Implantation of Artificial Cell Bioencapsulated Stem Cells to Increase the Survival of 90% Hepatectomized Rats

We implanted artificial cell bioencapsulated bone marrow mesenchymal stem cells into the spleens of 90% hepatectomized (PH) rats. The resulting 14 days survival rate was 91%. This is compared to a survival rate of 21% in 90% hepatectomized rats and 25% for those receiving free MSCs transplanted the same way. Unlike free MSCs, the bioencapsulated MSCs are retained in the spleens and their hepatotrophic factors can continue to drain directly into the liver without dilution resulting in improved hepatic regeneration. In addition, with time the transdifferentiation of MSCs into hepatocyte-like cells in the spleen renders the spleen as a ectopic liver support.

Effects of PEG-PLA-nano Artificial Cells Containing Hemoglobin on Kidney Function and Renal Histology in Rats

This study is to investigate the long-term effects of PEG-PLA nano artificial cells containing hemoglobin (NanoRBC) on renal function and renal histology after 1/3 blood volume top loading in rats. The experimental rats received one of the following infusions: NanoRBC in Ringer lactate, Ringer lactate, stroma-free hemoglobin (SFHB), polyhemoglobin (PolyHb), autologous rat whole blood (rat RBC). Blood samples were taken before infusions and on days 1, 7 and 21 after infusions for biochemistry analysis. Rats were sacrificed on day 21 after infusions and kidneys were excised for histology examination. Infusion of SFHB induced significant decrease in renal function damage evidenced by elevated serum urea, creatinine and uric acid throughout the 21 days. Kidney histology in SFHb infusion group revealed focal tubular necrosis and intraluminal cellular debris in the proximal tubules, whereas the glomeruli were not observed damaged. In all the other groups, NanoRBC, PolyHb, Ringer lactate and rat RBC, there were no abnormalities in renal biochemistry or histology. In conclusion, injection of NanoRBC did not have adverse effects on renal function nor renal histology.

Transplantation of bioencapsulated bone marrow stem cells improves hepatic regeneration and survival of 90% hepatectomized rats: a preliminary report.

We transplanted bioencapsulated bone marrow stem cells intraperitoneally into 90% hepatectomized rats and found that this increases both the rates of hepatic regeneration and survival of the animals. Bone marrow cells isolated from Wistar rats were bioencapsulated using alginate-polylysine-alginate method. These bioencapsulated bone marrow cells were transplanted intraperitoneally into 90% hepatectomized syngeneic wistar rats. Control groups included 90% hepatectomized group receiving intraperitoneal injection of either empty microcapsules or free bone marrow cells. Unlike the control groups, transplantation of bioencapsulated bone marrow cells improved the survival of 90% hepatectomized rats, with an efficacy similar to that of bioencapsulated hepatocytes or free hepatocytes. These results suggest that syngeneic bioencapsulated bone marrow stem cells can increase the survival rates of 90% hepatectomized rats. We also discuss the potential for a new alternative to hepatoctye transplantation for cellular therapy of acute liver failure. In particular, bone marrow stem cells can be obtained from the same patient with no immunorejection, whereas in hepatocyte transplant, immunosuppressant will be needed to prevent immunorejection of the donor hepatocytes.

Intrasplenic Transplantation of Bioencapsulated Mesenchymal Stem Cells Improves the Recovery Rates of 90% Partial Hepatectomized Rats

Mesenchymal stem cells (MSCs) derived from bone marrow can secrete cytokines and growth factors and can transdifferentiate into liver cells. We transplanted polymeric membrane bioencapsulated MSCs into the spleens of 90% partial hepatectomized rats. This resulted in 91.6% recovery rates. This is compared to a recovery rate of 21.4% in the 90% hepatectomized rats and 25% in the 90% hepatectomized rats receiving intrasplenic transplantation of free MSCs. After 14 days, the remnant livers in the bioencapsulated MSCs group are not significantly different in weight when compared to the sham control group. From day 1 to day 3 after surgery, in the bioencapsulated MSCs group, the plasma HGF and IL-6 were significantly higher than those in the free MSCs group and control group (P < 0.01); plasma TNF-α was significantly lower (P < 0.001). We concluded that the intrasplenic transplantation of bioencapsulated MSCs significantly increases the recovery rates of 90% hepatectomized rats. It is likely that the initial effect is from proliver regeneration factors followed later by the transdifferentiated hepatocyte-like cells. However, histopathological analysis and hepatocyte proliferation study will be needed to better understand the regenerative mechanisms of this result. This study has implications in improving the survival and recovery of patients with very severe liver failure due to hepatitis, trauma, or extensive surgical resection.