1. Transplanted adult hematopoietic stems cells differentiate into functional endothelial cells
by Alexis S. Bailey, Shuguang Jiang, Michael Afentoulis, Christina I. Baumann, David A. Schroeder, Susan B. Olson, Melissa H. Wong, and William H. Fleming
Blood
Volume 103(1):13-19
January 1, 2004
©2004 by American Society of Hematology

2. Bone marrow–derived cells differentiate into portal vein endothelium.
Bone marrow–derived cells differentiate into portal vein endothelium. Following transplantation of BM or HSCs (KSL), recipient liver was examined for donor-derived CD31+/VWF+ cells. (A) Hematoxylin and eosin stain of recipient liver tissue showing the lumen of a portal vein (L). (B) Y-chromosome–positive (red) male donor cell in the nucleus of a cell in the intima of a portal vein 8 months after KSL transplantation detected by Y-PAINT/FISH with DAPI (blue) nuclear staining. (C) X-gal detection of LacZ expression in a ROSA26 donor-derived cell (blue) in a portal vein. (D-F) A portal vein from a GFP+ KSL transplant recipient demonstrating the expression of CD31 (red, D) and GFP (green, E). (F) Merged image. (G-I) A portal vein from a GFP+ KSL recipient demonstrating the expression of VWF (red, G) and GFP (green, H). (I) Merged image. (J) Confocal microscopy images of a donor-derived GFP+, CD31+, DAPI+ cell in a portal vein; (K) a GFP+, VWF+, DAPI+ cell in a portal vein. (B,J-K, 500 KSL cells per recipient; C, 106 BM cells per recipient; D-F,G-I, 2000 KSL cells per recipient. Scale bars: A, D-I, 20 μm; B-C, 10 μm; J-K, 5 μm; L indicates portal vein lumen.)‏
Alexis S. Bailey et al. Blood 2004;103:13-19
©2004 by American Society of Hematology

3. HSCs give rise to circulating hematopoietic cells and functional ECs
HSCs give rise to circulating hematopoietic cells and functional ECs. The liver of recipients that received transplants of 500 GFP+ KSL cells was evaluated.
HSCs give rise to circulating hematopoietic cells and functional ECs. The liver of recipients that received transplants of 500 GFP+ KSL cells was evaluated. (A) GFP+ KSL cell–derived blood cells within a longitudinal section of a hepatic vessel (lumen (L) outlined, FITC channel only). (B) Merged image showing donor, GFP+ (green), DAPI+ (blue) nucleated blood cells coexpressing CD45 (red) and a single host-derived GFP–, CD45+ blood cell (arrow). (C) A portal vein with a donor DAPI+, GFP+, CD45– EC (arrowhead) near a donor DAPI+, GFP+, CD45+ blood cell (arrow). (D) Low-density lipoprotein (Dil-Ac-LDL, red) uptake in GFP+ ECs. Insert shows higher magnification of a single GFP+, Dil-Ac-LDL cell (arrow head). (E) A GFP+, CD45–, DAPI+ EC (arrowhead) within a portal vein from a 500 GFP+ KSL recipient 2 weeks after transplantation not injected with ac-LDL. Scale bars: A-B, 10 μm; C, 5 μm; and D-E, 20μm; original magnification, × 60 (D-E inserts).
Alexis S. Bailey et al. Blood 2004;103:13-19
©2004 by American Society of Hematology

4. Analysis of the DNA content of donor-derived hematopoietic cells and ECs in recipients of 500 KSL cells.
Analysis of the DNA content of donor-derived hematopoietic cells and ECs in recipients of 500 KSL cells. Top left panel shows GFP+ donor-derived cells in a recipient liver by flow cytometry. Top right panel indicates cell sorting gates for GFP+, CD31+, CD45– ECs and GFP+, CD31+, CD45+ hematopoietic cells. Sorted cell populations were fixed with ethanol and analyzed for DNA content with propidium iodide. Lower panels show propidium iodide uptake by sorted GFP+, CD31+, CD45– ECs, GFP+, CD31+, CD45+ hematopoietic cells, and control normal spleen cells. The percentage of cells in S/G2/M of the cell cycle is indicated.
Alexis S. Bailey et al. Blood 2004;103:13-19
©2004 by American Society of Hematology

5. High levels of donor-derived ECs and hematopoietic cells following primary and secondary transplantation.
High levels of donor-derived ECs and hematopoietic cells following primary and secondary transplantation. (A) Liver from recipients of GFP+ BM (2-5 × 106 cells), GFP+ KSL ( cells), or from unfractionated bone marrow (WBM) (106 cells) serially transplanted from primary recipients (secondary transplantation) was examined to determine the frequency of portal veins with GFP+ ECs. Portal veins were considered positive if 10% or more of the ECs integrated within the vessel wall were GFP+, CD31+, or VWF+. 2nd Tx indicates secondary transplantation. (Error bars indicate SEM; n = 3-6 mice per group [P ≤ .01]). (B) Donor-derived, multilineage hematopoiesis in the peripheral blood of a secondary recipient 3 months after transplantation. The percentage of GFP+ donor cells expressing individual hematopoietic lineage markers is indicated.
Alexis S. Bailey et al. Blood 2004;103:13-19
©2004 by American Society of Hematology

6. Bone marrow–and HSC-derived ECs are detected in multiple tissues.
Bone marrow–and HSC-derived ECs are detected in multiple tissues. (A) GFP+ (green) KSL-derived CD31+ (red) cell (arrow) and a GFP+, VWF+ (red) cell (insert) in skeletal muscle. (B) GFP+ BM-derived CD31+ cells in lung (arrow; insert: high magnification; A indicates alveolus, L indicates vascular lumen). (C) GFP+ BM-derived CD31+ cells in the distal small intestine. Donor-derived gut-associated lymphatic tissue is indicated (arrowheads) and a cross-section of a GFP+ CD31+ vessel is shown (arrow; insert: high magnification of neighboring GFP+ vessel). (D) GFP+ KSL-derived VWF+ cells in ventricular free wall of the heart (arrow; insert: higher magnification; V indicates ventricular cavity). (A,D, 2000 KSL cells; B-C, 106 BM. A-B, DAPI nuclear stain (blue). Scale bars: A, 10 μm; B, 20 μm; C, 25 μm; and D, 10 μm.) Original magnifications: × 40 (A,C inserts); × 60 (B,D inserts).
Alexis S. Bailey et al. Blood 2004;103:13-19
©2004 by American Society of Hematology

7. Transplantation of a single HSC produces multilineage hematopoiesis in the peripheral blood of recipient mice.
Transplantation of a single HSC produces multilineage hematopoiesis in the peripheral blood of recipient mice. Top panels: Isolation of single GFP+ KSL cells. (Primary and secondary cell sorting gates are shown). Middle panel: Donor-derived cells in the peripheral blood of a recipient of a single GFP+ KSL cell. Lower panels: GFP+ peripheral blood myeloid cells (GR-1) and B cells (B220) derived from a single KSL cell.
Alexis S. Bailey et al. Blood 2004;103:13-19
©2004 by American Society of Hematology

8. Transplantation of a single HSC gives rise to donor-derived ECs
Transplantation of a single HSC gives rise to donor-derived ECs. (A) Portal vein from a recipient of a single GFP+ KSL cell 6 months after transplantation demonstrating the expression of CD31 (Cy3, red channel only).
Transplantation of a single HSC gives rise to donor-derived ECs. (A) Portal vein from a recipient of a single GFP+ KSL cell 6 months after transplantation demonstrating the expression of CD31 (Cy3, red channel only). (B) High magnification of area in the box indicated in panel A, showing a GFP+ luminal cell (green channel only) integrated into the vessel wall. (C) Merged image, the box indicating the same donor GFP+ (green), CD31+ (red) EC with a DAPI+ nucleus (blue) (insert: high magnification of the same cell). (D) A GFP+, CD45– EC within the vessel wall (solid arrow) and a CD45+ (red) leukocyte (open arrowhead). (E) The same GFP+, CD45– EC sequentially labeled with VWF (red) (solid arrow). (F) A GFP+, CD45– donor EC in a liver venous sinusoid (solid arrow) and a donor CD45+ (red) leukocyte (open arrowhead) 6 months after transplantation. (G) The same GFP+ CD45– EC sequentially labeled with CD31 (red). (Scale bars: A, 20 μm; C: 20 μm; and D-G: 10 μm; DAPI+ nuclei (blue); L indicates lumen.) Original magnification, × 60 (B,C insert).
Alexis S. Bailey et al. Blood 2004;103:13-19
©2004 by American Society of Hematology