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. 2014 Jul 10;3(2):324–338. doi: 10.1016/j.stemcr.2014.06.005

Figure 3.

Figure 3

Fetal Keratinocytes Have a Higher Proliferation Potential than Adult Keratinocytes

(A) Proliferation index (PI) in fetal and adult epidermis. The PI was defined as the number of Ki67-positive cells divided by the total number of cells at the basal layer × 100%. Data are represented as mean ± SEM of n biological replicates.

(B) Comparison of the proliferation rates of fetal keratinocytes (17, 19, and 22 weeks) and adult keratinocytes. Data were generated by counting the number of cells after each passage, subcultured at 70%–80% confluence.

(C) Population doubling (pd) times are derived from each exponential phase of the growth curves monitored by a real-time cell analyzer (see Figure S5). Fetal (14, 16, 19, and 22 weeks) and adult keratinocytes were plated in 96-well plates at 2,500 cells per well. Cell growth was monitored over a period of 1 week. Data are represented as mean ± SD of n biological replicates. ∗∗∗∗p < 0.0001.

(D) Mean telomere restriction fragment (TRF) length of fetal (14 weeks) and adult keratinocytes. DNA (2 μg) prepared from keratinocytes was digested with Hinf I and Rsa I, and then separated on a 0.9% agarose gel by gel electrophoresis. It was then transferred to nylon, probed with a Dig-labeled telomere probe (TTAGGG), and detected via chemiluminescence. The average TRF length was determined by comparing the location of the TRF on the blot relative to a molecular weight standard. Fetal keratinocytes have longer telomeres than adult keratinocytes. In both adult and fetal keratinocytes, telomeres shorten with increasing passage number.

(E) Comparison of colony-forming activity between cultured fetal (17, 19, and 22 weeks) and adult keratinocytes. The colony-forming efficiency (CFE) was defined as the percentage of colonies formed over the number of cells seeded. A colony was defined as a cluster of >1 mm2. After 14 days, culture was arrested and the colonies were stained with Rhodamine B. Fetal keratinocytes form more colonies than adult keratinocytes. Data are represented as mean ± SD of three technical replicates.