Figure 1.
EPC-CFA profiles of HUCB CD34+ cells and mixed CD34+ and CD34− cells.
(A) Morphology of small EPC-CFUs and large EPC-CFUs derived from HUCB CD34+ cells (Scale bar = 20 µm). (B) EPC differentiation and expansion potentials were estimated by quantification of the 2 types of EPC-CFU clusters in CD34+ cell-derived EPC-CFUs and mixed CD34+ and CD34− cell-derived EPC-CFUs. The results are shown as means ± SEM (***p<0.001 vs. CD34+ cell-derived EPC-CFUs). (C) Endothelial characteristics of CD34+ cell-, CD34− cell-, and mixed CD34+ and CD34− cell-derived EPC-CFUs were assessed for Ac-LDL uptake (red) and Ulex europaeus agglutinin I (UEA-1)-conjugated FITC binding (green) (Scale bar = 100 µm). (D) Standard quantification of endothelial characteristics of EPC-CFUs was performed by counting the number of double-stained cells. The results are shown as means ± SEM (**p<0.01 vs. CD34+ cell-derived EPC-CFUs). CD34− cells could not be differentiated into either type of EPC-CFU clusters.
Figure 2.
Assessment of EPC differentiation and expansion potentials according to the number of HUCB CD34+ cells.
EPC differentiation and the expansion potential were investigated after coculture of varying densities of CD34+ cells (5, 100, 250, 500, 750, and 1000 cells per 35-mm dish) with CD34− cells (1000 cells per 35-mm dish). Standard quantification of EPC-CFUs was performed by counting the number of small, large, and total EPC-CFUs. (A) The number of the small EPC-CFUs was not significantly different among the groups. (B and C) Large EPC-CFUs and total EPC-CFUs were significantly increased in the CD34+-CD34− cell coculture compared to that in CD34+ cell culture alone condition. The results are shown as means ± SEM (*p<0.05 and **p<0.01 vs. CD34+ cell-derived EPC-CFUs).
Figure 3.
Optimal proportion CD34+ cells to CD34− cells for effective EPC differentiation.
The optimal proportion of CD34+ cells to CD34− cells was estimated by coculture of CD34+ cells (500 cells per 35-mm dish) with various densities of CD34− cells (100, 50, 20, 10, 5, 2.5, 1.7, 1.25, 1, 0.3, and 0% of CD34+ cells). (A) Small EPC colonies were not significantly increased in all groups. (B and C) Large EPC-CFUs and total EPC-CFUs were significantly increased in coculture of CD34+ and CD34− cells compared with CD34+ cell culture only in several groups. The results are shown as means ± SEM (*p<0.05 and **p<0.01 vs. CD34+ cell-derived EPC-CFUs).
Figure 4.
Assessment of the function of EPC-CFUs in response to CD34− cells.
(A) A schematic diagram to evaluate the effect of CD34− cells on the expansion of CD34+ cells using a transwell culture system. (B) Standard quantification of the expansion of CD34+ cells was performed by calculating the number of expanded CD34+ cells (lower chamber) using transwell cultures with or without CD34− cells (upper chamber). The results are shown as means ± SEM (**p<0.01 vs. transwell culture of CD34+ cells without CD34− cells). (C) Adhesion capacity was estimated by culture on human fibronectin-coated well plates. Standard quantification of adhesion cells was analyzed by the Cell Counting Kit-8 with detection at 490 nm using a microplate reader. The results are shown as means ± SEM (**p<0.01 vs. CD34+ cell-derived EPC-CFUs). (D) Tube formation capacity was assessed by tube formation assay of HUVECs cocultured with EC culture medium, CD34+ cells, mixed CD34+ and CD34− cells, conditioned medium from cultured CD34+ cells, and conditioned medium from cocultured CD34+ and CD34− cells in Matrigel. The results are shown as means ± SEM (*p<0.05, **p<0.01, and ***p<0.001 vs. each group). (E) Migration was determined by the Boyden chamber assay with HUVECs treated with serum- and growth factor-free EC medium, complete EC medium, and complete EC medium containing CD34− cells for 24 h. Migrated cells were counted in 5 random microscopic fields in the lower chamber by a hemocytometer with Cell Counting Kit-8 with detection at 490 nm using a microplate reader. The results are shown as means ± SEM (**p<0.01 vs. the complete EC medium-treated group).
Figure 5.
Assessment of the functional recovery in murine hind-limb ischemia.
(A) Laser Doppler perfusion imaging (LDPI) analysis of murine hind-limb ischemia transplanted with PBS, CD34+ cell-derived large EPC-CFUs (CD34(+)), and mixed CD34+ and CD34− cell (1.25% CD34+ cells with CD34− cells)-derived large EPC-CFUs (Hybrid) on days 0 and 28 post surgery. (B) Blood flow recovery ratio obtained by dividing the blood flow of the ischemic (left) limb by that of the nonischemic (right) limb. LDPI was measured on days 0 and 28 post operation. The results are shown as means ± SEM (**p<0.01 vs. the PBS-transplanted group). (C) At postoperative day 28, ischemic limb tissues were analyzed for the formation of capillary by staining for anti-CD31 (red) antibodies. (D) Standard quantification of the capillary density was evaluated by counting CD31+ cells per high-power field (hpf). The results are shown as means ± SEM (**p<0.01 vs. the PBS-transplanted group). (E) At postoperative day 28, ischemic limb tissues were analyzed for the engraftment of large EPC-CFUs into CD31-positive vessels by staining for anti-HNA (red) and anti-CD31 (green) antibodies. (F) Standard quantification of the engraftment of large EPC-CFUs into CD31-positive vessels was evaluated by counting CD31 and HNA-positive cells per hpf. The results are shown as means ± SEM (**p<0.01 vs. the CD34+ cell-derived large EPC-CFUs transplanted group).
Figure 6.
Identification of hematopoietic cell populations that regulate EPC development from HUCB CD34+ cells.
Standard quantification of small EPC colonies (A) and large EPC colonies (B) was performed by counting the number of each type of colony cluster after EPC-CFA of HUCB CD34+ cells cocultured with hematopoetic cells (HUCB CD34−) which consist of various cells such as macrophages (CD11b+ and CD11b− cells), T cells (CD3+ and CD3− cells), B cells (CD19+ and CD19− cells), megakaryocytes (CD41+ and CD41− cells), and monocytes (CD14+ and CD14− cells) on CD34− cells that were isolated using FACS analysis for specific markers from CD34− cells. Control represents each number of colony clusters of HUCB CD34− cells, CD34+ cells, or coculture of CD34+ and CD34− cells. The other bars, macrophage, T cell, B cell, megakaryocte, and monocyte represents each number of colony clusters of HUCB CD34+ with CD 11b− or with CD 11b+, with CD3− or with CD3+, with CD19− or with CD19+, with CD41− or with CD41+, and with CD14− or with CD14+, respectively. The results are shown as means ± SEM (**p<0.01 vs. culture of CD34+ only or coculture of CD34+ and various negative cells such as CD11b−, CD3−, CD19−, CD41−, or CD14− cells, respectively).
Figure 7.
Schematic model of the status of EPC development through specific cross talk with key hematopoietic cells isolated from CD34− cells.
EPC-CFA identifies the EPC differentiation hierarchy, early/small EPCs (primitive EPCs), and late/large EPCs (definitive EPCs) by the morphological and functional hallmarks. In EPC-CFA, CD34− cells, in particular T lymphocytes, macrophages, monocytes, and megakaryocytes, play a pivotal role in EPC development. T lymphocytes activate the differentiation CD34+ cells into primitive EPCs, which are round-shaped and proliferative. Macrophages, monocytes, and megakaryocytes accelerate the differentiation CD34+ cells and/or primitive EPCs into definitive EPCs, augmenting the adhesion, tube formation, and migration capacity, and enhancing the functional recovery, blood flow recovery, and capillary formation capacity, in vivo.