Transplantation of Bioencapsulated Bone Marrow Stem Cells Improve Hepatic Regeneration and Survival of 90% Hepatectomized Rats: A Preliminary Report

Abstract

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.

Keywords:

• Cell encapsulation
• Hepatectomy
• Stem cells
• Hepatocytes
• Liver failure
• Bone marrow cells
• Transdifferentiation
• Transplantation
• Artificial cells
• Hepatic regeneration

INTRODUCTION

Cell bioencapsulation is the enclosure of cells inside polymeric membrane microcapsules for the immunoprotection of transplanted cells, and the term “artificial cells” was introduced to define the concept of bioencapsulation [[1], [2]]. Transplantation of bioencapsulated hepatocytes was effective for experimental liver failure induced either by partial hepatectomy (PH) or by galactosamine [[3-7]]. Our previous studies showed cobioencapsulated bone marrow cells (BMCs) and hepatocytes improved the viability and function of hepatocytes in vivo and in vitro [[8-10]]. The use of D-galactosamine induced liver failure rats for studying the effects of liver regeneration and survival [[11]] is subject to difficulties in interpretation because of the complexity of this model. We therefore use a 90% partial hepatectomy rat model for the present study since the result can be more conclusively analyzed. Using this rat model, we compared the effects of intraperitoneal injection of free bone marrow stem cells to that of bioencapsulated bone marrow stem cells. We also compare this to the intraperitoneal injection of free hepatocytes or bioencapsulated hepatocytes.

MATERIALS AND METHODS

Animals

Donors of BMCs and hepatocytes were male Wistar rats, 200–225 g, purchased from Charles River (St-Constant, Canada). Recipients were the syngeneic male Wistar rats undergoing 90% PH. Animal study protocol was approved by the ethnic committee of McGill University.

BMCs and Hepatocytes Isolation

Wistar rats were anaesthetized with sodium pentobarbital and both femurs were isolated. Iscove's Modified Dulbecco's Medium (IMDM, GIBCOBRL, Life Technologies, NY) was used to flush out BMCs from the femurs using a 5 ml syringe with a 22 gauge needle. Hepatocytes were isolated using the method described by Seglen [[12]]. After perfused with collagenase buffer, the liver was excised and minced in cold William's E medium (GIBCOBRL, Life Technologies, NY) and shaken to free the hepatocytes. The hepatocytes were collected and filtered through a nylon sieve (85 µm), then washed with William's E medium and centrifuged at 50 G for 5 minutes at 4°C.

BMCs and Hepatocytes Encapsulation

Alginate-Polylysine-Alginate (APA) encapsulation method was used to encapsulate the BMCs or hepatocytes as described previously [[8]]. Nucleated BMCs 2 × 10⁸ were suspended in 25 ml 1.5% sodium alginate solution (Inotech, Rockville, USA); the cell suspension was extruded through droplet generator NISCO Encapsulator (NISCO Engineering AG, Switzerland).

For the hepatocytes encapsulation, 1 × 10⁸ hepatocytes in William's E medium were washed with buffered saline, and suspended in 15 ml of 1.5% (w/v) sodium alginate, and then encapsulated as for the BMCs encapsulation using NISCO Encapsulator as described above. The final hepatocytes microcapsules were placed in William's E medium and put in 5% CO₂, 37°C incubator for recovery before transplantation.

Ninety Percent Hepatectomy

Ninety percent partial hepatectomy was performed on Wistar male rats. Rats were anaesthetized with sodium pentobarbital, a 2-cm abdomen midline incision was made, the left and median lobes were isolated and pedicles ligated and resected. Then the lower and upper parts of the right lobe were isolated, ligated and resected, leaving only the caudate lobe [[13]]. For the Sham operation, we only carried out abdomen incision and cutting of the suspending ligament of the liver, and then closed the incision.

Transplantation Protocol

Immediately after 90% partial hepatectomy (PH), 42 rats were divided into 7 groups as follows: ([1]) PH only; ([2]) sham surgery; ([3]) PH with transplantation of 2 ml empty capsules; ([4]) PH with transplantation of 3 × 10⁷ bioencapsulated BMCs; ([5]) PH with transplantation of 3 × 10⁷ free BMCs; ([6]) PH with transplantation of 3 × 10⁷ bioencapsulated hepatocytes; [[7]] PH with transplantation of 3 × 10⁷ free hepatocytes. The animals were followed for 14 days.

Statistical Analysis

X² method was used for animal survival rate study. A p-value of less than 0.05 was regarded as significant.

RESULTS

Viability of Isolated Cells

Two femur bones in each rat yielded approximately 3 × 10⁸ nucleated BMCs. BMCs' viability after bioencapsulation was 98%. Hepatocytes' viability after encapsulation was 82%.

Animal Survival

The animal survival rate for each group is shown in Table 1. The survival rate was 100% in the group receiving bioencapsulated BMCs and also in the sham control group. In the two groups receiving bioencapsulated hepatocytes or free hepatocytes, the survival rate was also not significantly different from that of the sham control group. For those groups that received free BMCs, empty capsules or PH alone, the survival rates were significantly lower than the sham control group, with most of the mortality occurring in the first 3 days post-surgery (Table 1).

Table 1. Survival rate (%): (1) PH alone; (2) Sham surgery; (3) PH & empty capsules; (4) PH & encapsulated BMCs; (5) PH & free BMCs; (6) PH & encapsulated hepatocytes; (7) PH & free hepatocytes (*p < 0.05, **p < 0.01 versus sham surgery group)

	Groups
Day(post-surgery)	1	2	3	4	5	6	7
0	100	100	100	100	100	100	100
1	50*	100	83	100	83	100	100
2	17**	100	33**	100	50*	100	83
3	17**	100	33**	100	30**	83	83
7	17**	100	33**	100	17**	83	83
14	17**	100	33**	100	17**	83	83

The results of detailed histological studies and biochemical studies to be reported elsewhere correspond to the above observation.

Discussion

In this liver failure model, most deaths occur in the first 3 days after 90% hepatectomy; if animals survive after this critical period, deaths rarely occur. Our present study shows that intraperitoneal injection of bioencapsulated bone marrow stem cells (BMCs) can improve the survival rates in the 90% PH rat model. The effectiveness is not significantly different from free or bioencapsulated hepatocytes. However, unlike bioencapsulated BMCs, free BMCs are not effective in improving survival rates. Our studies show that intraperitoneally transplanted free BMCs are not retrievable from the peritoneal cavity. On the other hand, bioencapsulated BMCs remain in the peritoneal cavity and can be retrieved two weeks after transplantation. Our thesis is that humoral factors secreted by the bioencapsulated BMCs stimulate the regeneration of the remnant liver in the first several days post transplantation. This is made possible by the use of bioencapsulated BMCs since they are found to remain in the peritoneal cavity during the 14 days. This way, humoral factors secreted are carried by the drainage from the peritoneal cavity to reach the liver via the portal circulation. Unlike bioencapsulated BMCs, free BMCs are rapidly removed from the peritoneal cavity and any secreted humoral factors would not reach the liver by the portal circulation.

This preliminary study shows that intraperitoneal transplantation of bioencapsulated syngeneic BMCs can increase the survival rate of 90% hepatectomy liver failure rats. This effectiveness is similar to syngeneic hepatocytes transplantation. This suggests a possible alternative to hepatocytes transplantation for the treatment of liver failure. This is particularly exciting since hepatocytes have to be obtained from donors and thus requiring immunosuppression. On the other hand, BMCs can be obtained directly from the bone marrow of the same patient and thus do not need immunosuppressant. Further studies are needed to investigate the BMCs transdifferention and the mechanism of therapeutic effect. Further details will be reported elsewhere.

Operating Grant (MOP-13745) to TMS Chang (PI) from the Canadian Institutes of Health Research.