Sphere-forming corneal cells repopulate dystrophic keratoconic stroma: Implications for potential therapy

doi:10.4252/wjsc.v12.i1.35

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Jan 26, 2020 (publication date) through Apr 25, 2024

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World Journal of Stem Cells

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1948-0210

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Baishideng Publishing Group Inc, 7041 Koll Center Parkway, Suite 160, Pleasanton, CA 94566, USA

Basic Study

World J Stem Cells. Jan 26, 2020; 12(1): 35-54
Published online Jan 26, 2020. doi: 10.4252/wjsc.v12.i1.35

Table 1 PrimeTime quantitative polymerase chain reaction assays used for droplet digital polymerase chain reaction

Product of interest: Gene	Reference gene or gene of interest	Assay identification	Size of complimentary DNA amplicon, base pairs
Actin, beta	Reference	Hs.PT.39a.22214847	109
Beta-2-microglobulin	Reference	Hs.PT.58v.18759587	142
Glucuronidase beta	Reference	Hs.PT.58v.27737538	127
Glycreraldehyde-3-phosphate dehydrogenase	Reference	Hs.PT.39a.22214836	142
Hypoxanthine phosphoribosyltransferase 1	Reference	Hs.PT.58v.45621572	148
Peptidylprolyl isomerase A	Reference	Hs.PT.58v.38887593. g	101
Actin, alpha 2, smooth muscle, aorta	Gene of interest	Hs.PT.56a.2542642	131
ATP binding cassette subfamily B member 5	Gene of interest	Hs.PT.58.38502346	106
ATP-binding cassette, sub-family G member 2	Gene of interest	Hs.PT.58.20889058	114
Collagen type I, alpha 1 chain	Gene of interest	Hs.PT.58.15517795	130
Collagen type I, alpha 2 chain	Gene of interest	Hs.PT.58.26714160	108
Integrin, subunit beta 1	Gene of interest	Hs.PT.58.39883300	141
Keratin 3	Gene of interest	Hs.PT.58.25330614	126
Keratocan	Gene of interest	Hs.PT.58.45594658	130
Laminin, subunit alpha 1	Gene of interest	Hs.PT.58.3170022	129
Notch1	Gene of interest	Hs.PT.58.23074795	112
Proliferating cell nuclear antigen	Gene of interest	Hs.PT.58.4761611	94
Tumour protein p63, transcript variant 1, C-terminal isoform α	Gene of interest	Hs.PT.58.39019253	108
Tumour protein p63, transcript variant 1, N-terminal isoform	Gene of interest	Hs.PT.58.19710922	104
Vimentin	Gene of interest	Hs.PT.58.38906895	141

Table 2 Statistical analysis of difference in gene expression in sphere-seeded tissue sections as measured by droplet digital polymerase chain reaction, effect of time: day 0 vs day 14

	Day 0	Day 14	Day 14 - Day 0
Target	Estimate¹ (lower.95, upper.95)	Estimate¹ (lower.95, upper.95)	P
ABCB5	0.02 (0.01, 0.08)	0 (0, 0.01)	0.003	S
ABCG2	0.71 (0.25, 1.99)	0.02 (0.01, 0.05)	0.000	S
aSMA	0.21 (0.07, 0.58)	0.23 (0.09, 0.62)	0.871	NS
Col1A1	4.66 (1.65, 13.12)	4.97 (1.84, 13.44)	0.929	NS
Col1A2	11.33 (4.02, 31.92)	3.89 (1.44, 10.52)	0.145	NS
dNp63	0.03 (0.01, 0.09)	0 (0, 0)	0.000	S
ITGa3	0.12 (0.04, 0.33)	0.28 (0.10, 0.75)	0.244	NS
ITGb1	5.81 (2.06, 16.36)	7.99 (2.96, 21.59)	0.663	NS
Keratocan	0.14 (0.05, 0.39)	0.01 (0, 0.03)	0.000	S
KRT3	0.28 (0.08, 0.96)	0 (0, 0.01)	0.000	S
LAMA1	0.33 (0.12, 0.94)	0.07 (0.03, 0.19)	0.032	S
Notch1	0.43 (0.15, 1.21)	0.10 (0.04, 0.26)	0.043	S
P63a	0.05 (0.02, 0.15)	0 (0, 0.01)	0.000	S
PCNA	0.52 (0.19, 1.48)	0.71 (0.26, 1.92)	0.681	NS
Vimentin	16.96 (6.02, 47.77)	42.18 (15.61, 113.96)	0.214	NS

¹Values are the back-transformed estimates from the multivariate regression; NS: Not significant; S: Significant (P ≤ 0.05); ABCB5: ATP binding cassette subfamily B member 5; ABCG2: ATP-binding cassette, sub-family G member 2; αSMA: Smooth muscle, actin; COL1A1: Collagen type I, alpha 1 chain; COL1A2: Collagen type I, alpha 2 chain; TP63: Tumour protein p63; ∆NP63: Transcript variant 1, ∆NP63: N-terminal isoform, ITGa3: Integrin, subunit alpha 3; ITGB1: Integrin, subunit beta 1; KRT3: Keratin 3; LAMA1: Laminin, subunit alpha 1; TP63: Tumour protein p63; PCNA: Proliferating cell nuclear antigen.

Table 3 Statistical analysis of difference in gene expression in sphere-seeded tissue sections as measured by droplet digital polymerase chain reaction, effect of matrix, keratoconic vs normal

	Keratoconic	Normal	Keratoconic- normal
Target	Estimate¹ (lower.95, upper.95)	Estimate¹ (lower.95, upper.95)	P
ABCB5	0 (0, 0.01)	0.01 (0, 0.03)	0.351	NS
ABCG2	0.03 (0.01, 0.09)	0.37 (0.14, 0.99)	0.001	S
aSMA	0.24 (0.09, 0.64)	0.20 (0.07, 0.54)	0.800	NS
Col1A1	6.40 (2.37, 17.25)	3.62 (1.34, 9.77)	0.418	NS
Col1A2	9.30 (3.45, 25.08)	4.74 (1.76, 12.79)	0.337	NS
dNp63	0 (0, 0.01)	0.02 (0.01, 0.05)	0.004	S
ITGa3	0.16 (0.06, 0.43)	0.21 (0.08, 0.56)	0.695	NS
ITGb1	7.63 (2.83, 20.57)	6.09 (2.26, 16.42)	0.748	NS
Keratocan	0.03 (0.01, 0.08)	0.04 (0.02, 0.12)	0.588	NS
KRT3	0.01 (0.01, 0.04)	0.04 (0.01, 0.12)	0.211	NS
LAMA1	0.17 (0.06, 0.46)	0.13 (0.05, 0.36)	0.726	NS
Notch1	0.07 (0.03, 0.20)	0.58 (0.21, 1.56)	0.003	S
P63a	0.01 (0, 0.01)	0.02 (0.01, 0.06)	0.053	NS
PCNA	0.51 (0.19, 1.38)	0.73 (0.27, 1.97)	0.612	NS
Vimentin	24.29 (9.01, 65.52)	29.45 (10.92, 79.43)	0.784	NS
ABCB5	0 (0, 0.01)	0.01 (0, 0.03)	0.351	NS

Table 4 Viability of spheres cultured on keratoconic and normal tissue sections

Day post implantation	Keratoconic tissue sections		Normal tissue sections
	Number of sphere-implanted tissue sections cultured to time point	Number of sphere-implanted tissue sections viable at time point	Number of sphere-implanted tissue sections cultured to time point	Number of sphere-implanted tissue sections viable at time point
0	17		11
1	17	17	11	11
3	16	14	10	6
7	13	11	5	5
14	2	2

Citation: Wadhwa H, Ismail S, McGhee JJ, Van der Werf B, Sherwin T. Sphere-forming corneal cells repopulate dystrophic keratoconic stroma: Implications for potential therapy. World J Stem Cells 2020; 12(1): 35-54
URL: https://www.wjgnet.com/1948-0210/full/v12/i1/35.htm
DOI: https://dx.doi.org/10.4252/wjsc.v12.i1.35