Distinct contribution of human cord blood-derived endothelial colony forming cells to liver and gut in a fetal sheep model

Abstract

Although the vasculogenic potential of circulating and cord blood (CB)-derived endothelial colony-forming cells (ECFC) has been demonstrated in vitro and in vivo, little is known about the inherent biologic ability of these cells to home to different organs and contribute to tissue-specific cell populations. Here we used a fetal sheep model of in utero transplantation to investigate and compare the intrinsic ability of human CB-derived ECFC to migrate to the liver and to the intestine, and to define ECFC's intrinsic ability to integrate and contribute to the cytoarchitecture of these same organs. ECFCs were transplanted by an intraperitoneal or intrahepatic route (IH) into fetal sheep at concentrations ranging from 1.1-2.6 × 10(6) cells/fetus. Recipients were evaluated at 85 days posttransplant for donor (human) cells using flow cytometry and confocal microscopy. We found that, regardless of the route of injection, and despite the IH delivery of ECFC, the overall liver engraftment was low, but a significant percentage of cells were located in the perivascular regions and retained the expression of hallmark endothelial makers. By contrast, ECFC migrated preferentially to the intestinal crypt region and contributed significantly to the myofibroblast population. Furthermore, ECFC expressing CD133 and CD117 lodged in areas where endogenous cells expressed those same phenotypes.

Conclusion: ECFC inherently constitute a potential source of cells for the treatment of intestinal diseases, but strategies to increase the numbers of ECFC persisting within the hepatic parenchyma are needed in order to enhance ECFC therapeutic potential for this organ.

Figure 1. Human ECFC maintain DsRed expression and engraft preferentially in vascular and peri-vascular regions

(A) Representative image of the distribution of donor-derived ECFC in a liver section of transplanted animals. (B) Higher magnification demonstrating the vascular and peri-vascular engraftment of DsRed-positive ECFC. (C) Immunostaining of DsRed-positive ECFC (red) with an antibody against DsRed (green). Double-positive cells are shown in yellow. (D) Fluorescence in situ hybridization (FISH) of a chimeric liver tissue with a sheep-specific DNA probe labeling all nuclei except the human DsRed+ cells, demonstrating that these cells do not contain sheep DNA. (E) FISH of a chimeric liver tissue with a human-specific DNA probe demonstrating that only the human DsRed+ cells contain human DNA. The specificity of the human and sheep probes was determined using sheep and human liver tissue sections, respectively. (F). Following IH injection, 91.69%±3.12%(n=5) of the engrafted ECFCs were found in the vascular or peri-vascular regions of the fetal liver. IP injection resulted in significantly higher engraftment of ECFC in vascular and peri-vascular regions, 98.70%±0.92% (n=10), of the liver compared to IH injection. *= p<0.05, data shown as mean±SEM. Images were taken with an Olympus Fluoview 1000 Confocal system as described in the materials and methods section.

Figure 2. ECFC preferentially engraft in and around the crypts of Lieberkühn

(A) Low-magnification image of a representative section of the intestine of an animal transplanted with DsRed+ECFC; donor-derived cells preferentially engrafted in and around the crypts of Lieberkühn (CPT) (B) High magnification image of a representative section of the CPT intestinal region of an animal transplanted with DsRed+ECFC. (C) Overall engraftment vs CPT region engraftment in animals transplanted IP and IH. The preferential engraftment by the ECFC was conserved across transplanted animals, regardless of cell dose and injection route (* =p<0.05; *** = p < 0.001)

Figure 3. Engrafted ECFC continue to express markers of endothelial lineages

(A) Representative image of DsRed expression by the engrafted ECFC. (B) Immunostaining with human-specific antibody against CD31. (C) Merged image of DsRed+ cells showing co-localization with CD31. (D) Representative image of DsRed expression by the engrafted ECFC (E) Immunostaining with human-specific antibody against vWF. (F) Merged image of DsRed+ cells showing co-localization with vWF. Images were taken with an Olympus Fluoview 1000 Confocal system as described in the materials and methods section.

Figure 4. Engrafted ECFC express FVIII:c and CD34

(A) Representative image of DsRed expression by the engrafted ECFC. (B) Immunostaining with human-specific antibody against FVIII:c. (C) Merged image of DsRed+ cells showing expression of FVIII:c in some of the cells. (D) Representative image of DsRed expression by the engrafted ECFC. (E) Immunostaining with human-specific antibody against human CD34. (F) Merged image of DsRed+ cells showing co-localization with CD34. Images were taken with an Olympus Fluoview 1000 Confocal system as described in the materials and methods section.

Figure 5. ECFC express smooth muscle actin (SMA) and vimentin

(A) Donor-derived ECFC express SMA. (B) Donor-derived ECFC express vimentin. (C) Merged image of cells expressing SMA and vimentin. (D) Merged image of DsRed+ cells expressing both SAM and vimentin. (E) Detail from image D showing co-localization of DsRed+ cells expressing both SMA and vimentin. (F) Z-stack analysis of a 1μM thick stack of images confirms expression of both SAM and vimentin by a single donor-derived cell. Images were taken with an Olympus Fluoview 1000 Confocal system as described in the materials and methods section.

Figure 6. Donor-derived DsRed+ cells expressed CD117 and localized to interstitial areas where the endogenous CD117+ population could be found

(A) Engrafted DsRed+ cells. (B) Cells in the intestine expressing the interstitial cell marker CD117+. (C) Merged image of DsRed+ cells expressing CD117. (D) Z-stack analysis of a DsRed+ cell. (E) Z-stack analysis of a chromogranin A+ cell. (F) Z-stack analysis merged image of a DsRed+ that is expressing chromogranin A+. Images were taken with an Olympus Fluoview 1000 Confocal system as described in the materials and methods section.

Figure 7. Transplanted cells did not contribute to the intestinal epithelial cell layer and continue to express CD133

(A) Representative image of engrafted DsRed+ cells in the intestinal villi. (B) Cytokeratin 20 staining of the intestinal villi. (C) Merged image of DsRed+ cells in the tissue section stained with an antibody against Cytokeratin 20. (D) Representative image of engrafted DsRed+ cells in the CPT. (E) Cells in the intestine expressing the interstitial cell marker CD133. (F) Merged image of DsRed+ cells expressing CD133.