Human cord blood stem cells generate human cytokeratin 18-negative hepatocyte-like cells in injured mouse liver

Abstract

Differentiation of adult bone marrow (BM) cells into nonhematopoietic cells is a rare phenomenon. Several reports, however, suggest that human umbilical cord blood (hUCB)-derived cells give rise to hepatocytes after transplantation into nonobese diabetic-severe combined immunodeficient (NOD-SCID) mice. Therefore, we analyzed the hepatic differentiation potential of hUCB cells and compared the frequency of newly formed hepatocyte-like cells in the livers of recipient NOD-SCID mice after transplantation of hUCB versus murine BM cells. Mononuclear cell preparations of hUCB cells or murine BM from enhanced green fluorescent protein transgenic or wild-type mice were transplanted into sublethally irradiated NOD-SCID mice. Liver regeneration was induced by carbon tetrachloride injury with and without subsequent hepatocyte growth factor treatment. By immunohistochemistry and reverse transcriptase-polymerase chain reaction, we detected clusters of hepatocyte-like cells in the livers of hUCB-transplanted mice. These cells expressed human albumin and Hep Par 1 but mouse CK18, suggesting the formation of chimeric hepatocyte-like cells. Native fluorescence microscopy and double immunofluorescence failed to detect single hepatocytes derived from transplanted enhanced green fluorescent protein-transgenic mouse BM. Fluorescent in situ hybridization rarely revealed donor-derived hepatocyte-like cells after cross-gender mouse BM transplantation. Thus, hUCB cells have differentiation capabilities different from murine BM cells after transplantation into NOD-SCID mice, demonstrating the importance of further testing before hUCB cells can be used therapeutically.

Figure 1

Analysis of donor-derived epithelial cells in the livers of NOD-SCID mice transplanted with EGFP-positive BM mononuclear cells. a: Native fluorescence of one rare EGFP-positive nodule in an untreated tissue section. b: H&E staining of a representative section that was positive for EGFP in the native fluorescence microscopy. The EGFP-positive cells (arrow) are smaller in size and do not show morphological characteristics of hepatocytes. c–f: A rare GFP-positive nodule in one section was stained with GFP antibody, DPPIV antibody, and with DAPI together. Immunofluorescence staining for EGFP (c) and the canalicular marker DPPIV (d). e: The merged figure clearly demonstrates that this rarely found EGFP-positive nodule is not positive for DPPIV supporting the absence of hepatocyte-like cells in this nodule. f: DAPI staining of this nodule shows the distinct pattern of the nuclei in the EGFP-positive nodule compared to the surrounding endogenous hepatocytes. Original magnifications: ×20 (a, c–f); ×10 (b).

Figure 2

Mouse Y-chromosome FISH in gender-mismatched transplanted mice. a: Normal male mouse liver showing successful detection of Y-chromosome-positive nuclei. b: A rare Y-chromosome-positive nucleus in the mouse liver, transplanted with bone morrow cells of transgenic GFP mice (arrow). c: Normal female mouse liver showing complete absence of Y-chromosome. Original magnifications: ×63 (a); ×100 (b and c).

Figure 3

Effect of CCl₄ on human and mouse hepatocytes. a: AST and ALT were measured in supernatant by biochemical analyzer. Detoxification by CCl₄ was found to be similar because there is no significant difference between values of AST and ALT. b: Percentage survival of human and mouse hepatocytes by trypan blue exclusion test also demonstrates similar detoxification effect of CCl₄ on both cell types.

Figure 4

a–c: Detection of hUCB-derived cells in the liver of a NOD-SCID mouse after CCl₄ injury and HGF treatment by immunohistochemistry with the HepPar1 antibody. a: Human liver section was used as a positive control. b: Liver section from hUCB-transplanted NOD-SCID mice: single cells and small clusters of donor-derived cells stained positive for the human hepatocellular antigen Hep Par1 in hUCB-transplanted NOD-SCID mice (arrows). c: Sham-transplanted NOD-SCID liver ensures the complete absence of HepPar1-positive cells. d–f: Immunohistochemistry for human albumin in liver sections of NOD-SCID mice after hUCB BM reconstitution. d: Human liver sections were used as positive control. e: Single and small clusters of cells stained positive for human albumin in hUCB-transplanted NOD-SCID mice (arrows). f: Sham-transplanted NOD-SCID mouse liver. No human albumin-positive cell was detectable demonstrating the specificity of the antibody staining. g–i: Immunohistochemistry for CK18 expression in hUCB-transplanted NOD-SCID mice. g: In human liver the epithelial marker CK18 is strongly expressed by hepatocytes. h: Notably, CK18 expression was absent in hUCB-transplanted NOD-SCID mice suggesting an immature phenotype of the donor-derived cells. i: Liver sections of sham-transplanted NOD-SCID mice served as negative control. Original magnifications, ×40.

Figure 5

RT-PCR of human hepatocellular genes in hUCB-transplanted NOD-SCID mice after CCl₄ and HGF treatment. Lane 1: Liver tissue of sham-transplanted NOD-SCID mice served as negative control. Lane 2: Positive controls: cDNA from human liver for alb, CK18, and CK19, from HUH-7 human hepatoma cells for AFP. Lane 3: Liver tissue of hUCB-transplanted NOD-SCID mice. Specific PCR products were obtained with cDNA of transplanted NOD-SCID mouse liver for human albumin but not for CK18, CK19, and AFP. In addition to these markers, human CK8 and cytochrome P450 could not be detected in cDNA of transplanted NOD-SCID mouse liver (not shown). No PCR product was detected in the absence of reversed transcriptase (not shown). hALB, human albumin; hAFP, human α-fetoprotein; hCK18, human CK18; hCK19, human CK19; mβ-tub, mouse β-tubulin.

Figure 6

Immunostaining of serial sections reveals that hepatocyte-like cells were result of fusion (green, Hep Par 1; blue, human CK18; and red, mouse CK18). a–c: Serial sections of liver from hUCB-transplanted mouse. a: First serial section shows presence of donor-derived cells stained positive (arrow) for the human hepatocellular antigen Hep Par1, detected by Alexa Fluor 488 (green). b: Next section was stained for human CK18, which shows complete absence of human CK18. c: In third serial section all cells were found to be positive for mouse CK18. d: Human liver section stained for Hep Par 1 as a positive control. e: Human liver section stained for human CK18 as a positive control. f: DAPI staining of first serial section. g: Serial section was stained for human CK18, which also shows complete absence of human CK18, by immunohistochemical staining. h: H&E staining of one of these serial section. Original magnifications: ×10 (a–c, f–h); ×20 (d, e and insets in a–c).