Transplantation of human adipose tissue-derived multilineage progenitor cells reduces serum cholesterol in hyperlipidemic Watanabe rabbits

Abstract

Familial hypercholesterolemia (FH) is an autosomal codominant disease characterized by high concentrations of proatherogenic lipoproteins and premature atherosclerosis secondary to low-density lipoprotein (LDL) receptor deficiency. We examined a novel cell therapy strategy for the treatment of FH in the Watanabe heritable hyperlipidemic (WHHL) rabbit, an animal model for homozygous FH. We delivered human adipose tissue-derived multilineage progenitor cells (hADMPCs) via portal vein and followed by immunosuppressive regimen to avoid xenogenic rejection. Transplantation of hADMPCs resulted in significant reductions in total cholesterol, and the reductions were observed within 4 weeks and maintained for 12 weeks. (125)I-LDL turnover study showed that the rate of LDL clearance was significantly higher in the WHHL rabbits with transplanted hADMPCs than those without transplanted. After transplantation hADMPCs were localized in the portal triad, subsequently integrated into the hepatic parenchyma. The integrated cells expressed human albumin, human alpha-1-antitrypsin, human Factor IX, human LDL receptors, and human bile salt export pump, indicating that the transplanted hADMPCs resided, survived, and showed hepatocytic differentiation in vivo and lowered serum cholesterol in the WHHL rabbits. These results suggested that hADMPC transplantation could correct the metabolic defects and be a novel therapy for inherited liver diseases.

FIG. 1.

(A) Morphological characters of human adipose tissue-derived multilineage progenitor cells (hADMPCs). The cells obtained from adipose tissue were seeded and incubated for 24 h (i). After incubation, the adherent cells were treated with ethylenediaminetetraacetic acid solution, and the resulting suspended cells were replated at a density of 10,000 cells/cm² on human fibronectin-coated dishes (BD BioCoat) (ii, iii). Within two to three passages after the initial plating of the primary culture, hADMPCs appeared as a monolayer of large flat cells (25–30 μm in diameter). As the cells approached confluence, they assumed a more spindle-shaped, fibroblastic morphology (iv). i) Bar = 499 μm, ii) bar = 201 μm, iii) bar = 502 μm and iv) bar = 202 μm. (B) Cell surface markers expressed on hADMPCs. The cells were negative for markers of the hematopoietic lineage (CD45) and of hematopoietic stem cells, ABCG-2, CD34, and CD133. They were also negative for CD31, an endothelial cell-associated marker, and the surface antigen c-Kit (CD117). However, they stained positively for a number of surface markers characteristic of mesenchymal and/or neural stem cells, but not embryonic stem (ES) cells, including CD29, CD44 (hyaluronan receptor), CD73, CD105 (endoglin), and CD166. hADMPCs also were positive for stage-specific embryonic antigen (SSEA)-4. (C) Adipocytic, chondrocytic, and osteocytic differentiation potentials of hADMPCs. Adipocytic differentiation potential of hADMPCs was confirmed by Oil Red O staining (the left panel) (bar = 100 μm). Chondrocytic differentiation potential of hADMPCs was estimated by extracellular matrices with Alcian Blue staining (the middle panel). Osteogenic differentiation potential of hADMPCs was confirmed by Alizarin red S staining for mineralized nodules (the right panel).

FIG. 2.

(A) Immunosuppression regimen. Cyclosporin A (6 mg/kg/day) and rapamycin (0.05 mg/kg/day) were administered intramuscularly daily from the day before surgery to sacrifice. Methylprednisolone was administered at 3 mg/kg/day (days 1–7), 2 mg/kg/day (days 8–14), 1 mg/kg/day (days 15–21), and 0.5 mg/kg/day (day 22 to sacrifice). Cyclophosphamide (20 mg/kg/day) was injected intravenously at days 0, 2, 5, and 7. Ganciclovir (2.5 mg/kg/day) was also injected intramuscularly to avoid viral infection in the immunocompromised host. (B) Surgical procedure. Watanabe heritable hyperlipidemic (WHHL) rabbits were anesthetized with pentobarbital. An incision was made distal and parallel to the lower end of the ribcage. The peritoneum was incised and hADMPCs, and human adipose tissue-derived fibroblastic cells (hADFCs) (3 × 10⁷ cells/rabbit) or controls were infused into the portal vein using an 18-gauge Angiocath. (C) Localization of transplanted hADMPCs in the WHHL liver. At the day of and 1, 2, 4, 6, and 10 weeks after transplantation of DiI-labeled hADMPCs via the portal vein, the WHHL rabbit liver was examined histologically. DiI-fluorescent labeled-hADMPCs resided and distributed in the portal area at the day of transplantation. One to 2 weeks after transplantation, the DiI-stained hADMPCs-derived cells were localized near the portal areas. Four weeks after transplantation some of the DiI-stained cells resembled innate hepatocytes morphologically. Six and 10 weeks after transplantation, DiI-positive transplanted cells were dispersed in a centrilobular direction, resembling the mature innate hepatocytes. Bars = 100 μm. (D) Quantification of repopulation of the transplanted cells in the liver. The ratios of human-derived cell repopulation were examined by analyzing an Alu repetitive DNA sequence at the day of and 2, 4, 8, and 12 weeks after transplantation. In upper panel the standard curve was indicated, and in lower panel the ratio of repopulation of human cells was shown in time course after transplantation of hADMPCs.

FIG. 3.

(A) Total serum cholesterol levels. hADMPC transplantation in WHHL rabbits was followed for 12 weeks. Total serum cholesterol was measured in five rabbits that each received 3 × 10⁷ hADMPCs, three rabbits that each received 3 × 10⁷ hADFCs, and in six rabbits that received saline (control). Bars indicated mean ± standard error of the mean (SEM) (^#p < 0.05; control vs. the hADMPC-transplanted WHHL rabbit; ^&p < 0.05; the hADFC-transplanted WHHL rabbit vs. the hADMPC-transplanted WHHL rabbit). (B) Lipoprotein profiles in a representative WHHL rabbit with hADMPC transplantation after gel filtration. Serum samples from the WHHL rabbit before and 4 weeks after transplantation were fractionated. Note the marked reduction in low-density lipoprotein (LDL) peak and appearance of high-density lipoprotein (HDL) peak. (C) Rate of clearance of LDL from the serum of rabbits with and without transplantation of hADMPCs. Animals were injected with ¹²⁵I-labeled human LDL, and the time course of clearance was monitored following trichroloacetic acid precipitation of serum at time 5 min, 1 h, 2 h, 4 h, 6 h, and 28 h. Residual ¹²⁵I-LDL was expressed as percentages of that at 5 min. ^#p < 0.05 (control vs. the hADMPC-transplanted WHHL rabbit [low dose]) and *p < 0.05 (control vs. the hADMPC-transplanted WHHL rabbit [high dose]). (D) DiO-LDL uptake into hADMPC-derived hepatocytes in the WHHL rabbi liver. Thin-sliced recipient liver was incubated with DiO-labeled LDL in the serum-free medium for 24 h. After washing and fixation, the incubated slices were applied for fluorescent microscopy. DiO-LDL uptake cells (green) and no uptake parenchymal cells were observed in the section. Bar = 100 μm.

FIG. 4.

(A) Immunohistochemical identification of human hepatocytic marker cells in liver sections of WHHL rabbits after hADMPC transplantation. Twelve weeks after hADMPC transplantation, human albumin-, human alpha-1-antitrypsin-, human bile salt export pump (BSEP)-, and LDL-receptor-positive cells were dispersed within the perivenous regions of the liver parenchyma, where they made contact with and integrated among the host cells with cell–cell interactions between hADMPC-derived cells and diseased hepatocytes pair. Ten weeks after transplantation of DiI-stained hADMPCs, copresence of human albumin (green) and pretreated DiI-fluorescence (red) on the same cells was observed. Bar = 100 μm. (B) Differentiation of transplanted hADMPCs into hepatocyte-like cells. Twelve weeks after transplantation, almost but not all human CD90-positive cells expressed human albumin, indicating that major population of transplanted hADMPCs could differentiate into hepatocyte-like cells (left panel: human CD90; middle panel: human albumin; right panel: merge). Arrows indicate human CD90 and human albumin double-positive cells; arrowheads indicate human CD90-positive but human albumin-negative cells. (C) Human hepatic gene expression in WHHL rabbit liver after hADMPC transplantation. RNA was prepared from the WHHL rabbit liver 12 weeks after hADMPC transplantation. We used the following hepatic markers: human alpha-1-antitrypsin, human albumin, human factor IX, human GATA-binding protein 4 (GATA-4), human hepatocyte nuclear factor 3 (HNF-3) beta, and human LDL-receptor. Their expression levels were examined by quantitative real time-polymerase chain reaction (RT-PCR) using Assays-on-Demand Gene Expression Assay Mix. The livers of WHHL rabbits that received saline (n = 3) were negative for human hepatic genes. The mRNA levels were normalized based on human glyceraldehyde-3-phosphate dehydrogenase expression as housekeeping gene and data are mean ± SEM of triplicate experiments. The livers of WHHL rabbits that received hADMPC transplantation (n = 3) were positive for human hepatic genes, and their expression levels were similar to those of human primary hepatocytes but not hADMPCs per se. Data are mean ± SEM.