Angiogenesis potential of human limbal stromal niche cells - PubMed (original) (raw)
Comparative Study
Angiogenesis potential of human limbal stromal niche cells
Gui-Gang Li et al. Invest Ophthalmol Vis Sci. 2012.
Abstract
Purpose: The perivascular localization of stem cell (SC) niches suggests the presence of a vascular niche. We aimed to determine the angiogenesis potential of limbal niche cells (NCs).
Methods: Human limbal NCs were isolated and serially passaged on plastic or coated Matrigel in embryonic SC medium containing BFGF and leukemia inhibitory factor before being reseeded in 3D Matrigel. Expression of angiogenesis markers was assessed by RT-qPCR and immunofluorescence staining. Their angiogenesis potential was measured by differentiation into vascular endothelial cells and by supporting vascular tube network formed by human umbilical vein endothelial cells (HUVEC) on Matrigel. Their support of limbal epithelial progenitor cells (LEPC) was examined in sphere growth formed by reunion in 3D Matrigel.
Results: On plastic, limbal NC could be cultured only up to four passages before turning into myofibroblasts. In contrast, on coated Matrigel, they could be expanded for up to 12 passages with upregulation of markers suggestive of angiogenesis progenitors when reseeded in 3D Matrigel because they could differentiate into vascular endothelial cells and pericytes stabilizing the tube network formed by HUVEC. Although both expanded limbal NCs and HUVEC rejoined with LEPC to form spheres to upregulate expression of ΔNp63α, CK15, and CEBPδ, the former but not the latter abolished expression of CK12 keratin.
Conclusions: Human limbal NCs continuously expanded on the basement membrane differentiate into angiogenesis progenitors that prevent differentiation of LEPC/SCs. They may partake in formation of the vascular niche and contribute to angiogenesis during wound healing.
Conflict of interest statement
Disclosure: G.-G. Li, None; S.-Y. Chen, None; H.-T. Xie, None; Y.-T. Zhu, None; S.C.G. Tseng, None
Figures
Figure 1.
Serial passages on plastic. Cells isolated from collagenase-isolated clusters from a 62-year-old donor were serially passaged on plastic in ESCM containing LIF and BFGF. They yielded spindle cells (A) and could only reach P4 with a doubling time of more than 165 hours and NCD of 6 (B). When P3 single cells were reseeded in 3D Matrigel for 6 days, they generated P4/3D aggregates at Day 6 with a smooth contour (A). Compared with D0 cells just isolated, P3 spindle cells did not express Oct4, Sox2, Flk-1, CD34, CD31, PDGFRβ, and SMMHC transcripts, but expressed α-SMA and S100A4 transcripts (C, *P < 0.05, **P < 0.01). Furthermore, after being reseeded in 3D Matrigel, the resultant P4/3D cells did not regain expression of the above markers. Scale bar = 200 μm.
Figure 2.
Serial passages on coated Matrigel. Single cells derived from collagenase-isolated limbal clusters from one limbal segment of the same donor as in Figure 1 were serially passaged on coated Matrigel in ESCM with BFGF and LIF. They generated spindle cells from P1 to P12 (A) and had a steady proliferative rate with the doubling time of 43 to 47 hours from P2 to P10 (B). Bar = 100 μm.
Figure 3.
Pericyte phenotype promoted by serial passages on coated Matrigel. When compared with freshly isolated cells at D0, RT-qPCR revealed a notable decrease of Oct4, Sox2, Flk-1, CD34, CD31, SMMHC, and S100A4 transcripts but a dramatic increase of α-SMA and PDGFRβ transcripts during serial passages (A, n = 3, *P < 0.05, **P < 0.01). D0 cells consisted of PCK+ and Vim+ cells and expressed Oct4 and Sox2. In addition, Vim+ cells expressed Flk-1, CD34, CD31, or α-SMA (see photos with high magnification), but the overall percentage of colocalization was less than 1% (see photos with low magnification of double labeling of Flk-1/Vim, CD31/Vim and α-SMA/Vim, n = 1000), and none expressed PDGFRβ (B, D0). In contrast, P3 cells were all PCK-/Vim+, α-SMA+, and PDGFRβ+, but negative to Oct4, Sox2, Flk-1, CD34, and CD31 (B, P3). Nuclei were counterstained by Hoechst 33342 (blue). Scale bar = 50 μm.
Figure 4.
Angiogenesis progenitors promoted by reseeding in 3D Matrigel. P3 cells expanded on coated Matrigel were reseeded in 3D Matrigel, they formed P4/3D aggregates; single cells were collected on Day 6. Compared with P3 spindle cells, expression of Oct4, Sox2, Flk-1, CD34, CD31, α-SMA, and PDGFRβ transcripts were markedly upregulated in P4/3D cells (A, n = 3, *P < 0.05, **P < 0.01). In contrast, expression of SMMHC and S100A4 remained lacking. P4/3D aggregates were noted as early as 4 hours and exhibited with a stellate contour at Day 6 (B). Immunostaining of P4/3D cells showed uniform expression of Vim together with the above positive markers (B). Nuclei were counterstained by Hoechst 33342 (blue). Scale bar = 50 μm.
Figure 5.
Differentiation into vascular endothelial cells. Single cells of P4/3D aggregates were cultured on plastic in EGM2 with VEGF for 3 days, yielding spindle cells similar to HUVEC. They uniformly expressed Flk-1, CD31, and von Willebrand factor, and took up Dil-Ac-LDL (top) in a similar fashion to the positive control of HUVEC cultured in the same condition (bottom). Nuclei were counterstained by Hoechst 33342 (blue). Scale bar = 100 μm.
Figure 6.
Support of HUVEC vascular tube network. Fluorescence pre-labeled (red) HUVEC and P4/3D cells were seeded alone or together on the surface of 100% Matrigel in EGM2. Although vascular tube-like network was noted in all three conditions at Day 1 (A–C), such network in P4/3D cells (A) or HUVEC (B) alone was disintegrated by Day 2 (E, F). In contrast, the network formed by cocultured P4/3D cells and HUVEC (C) was maintained at Day 2 (G) and Day 5 (not shown). High magnification of insets (C, G) revealed close association between P4/3D cells and HUVEC (red) in the network at Day 1 (D) and Day 2 (H). Scale bar = 200 μm for A–C and E–G, and 50 μm for D and H.
Figure 7.
Epithelial sphere growth in 3D Matrigel. LEPCs derived from dispase-isolated epithelial sheets alone or mixed with fluorescence prelabeled (red) HUVEC or P4/3D cells to generate sphere growth from Day 2 to Day 10 in 3D Matrigel (A). Compared with those formed by LEPC alone, spheres formed by LEPC+HUVEC and by LEPC+P4/3D expressed significantly more ΔNp63α, CK15, and CEBPδ transcripts (B, n = 3, *P < 0.05, **_P_ < 0.01). In addition, expression of CK12 transcripts by LEPC+HUVEC was not different from that of LEPC alone (**B,** _n_ = 3, _P_ > 0.05), whereas that by LEPC+P4/3D was not detectable (B, n = 3, P < 0.01). Compared with LEPC alone, expression of p63α protein was elevated in both LEPC+HUVEC and LEPC+P4/3D (**C,** _n_ = 3, _P_ < 0.05). In contrast, expression of CK12 protein was not reduced in LEPC+HUVEC (**C,** _n_ = 3, _P_ > 0.05) but was reduced to a nondetectable level in LEPC+P4/3D using β-actin as a loading control (C, n = 3, P < 0.05). Double staining with p63α and CK12 showed that HUVEC or P4/3D cells alone did not expresss p63α or CK12, whereas CK12 was expressed by LEPC alone and LEPC+HUVEC but not LEPC+P4/3D (D). Scale bar = 200 μm for A and 100 μm for D.
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