Table 2 Frequency of ER^pos and lineage marker-positive cells as a function of passage number in culture.

Passage #	Per cent ER ⁺ cells	Per cent K8 ⁺ cells	Per cent K19 ⁺ cells	Per cent P63 ⁺ cells
2	41/21/24/38/42	100/100/100/ND/100	44/47/100/ND/60	0/5/0/ND/0
3	76^†/47/22/50^‡/26	100^†/100/100/100^‡/100	100^†/41/62/100^‡/76	0^†/2/0/0^‡/0
4	77^†/26^†/28/51^†/23	100^†/100^†/100/100^†/100	100^†/79^†/34/100^†/76	0^†/0^†/0/0^†/0
5	29^†/72^†/39/38/70/37	100^†/100^†/100/ND/ND/100	79^†/100^†/48/ND/29/99	0^†/0^†/0/ND/0*/6
6	66^†/27/42/22	100^†/100/ND/97	100^†/60/ND/69	0^†/0*/ND/8
7	54^†/28/30/25/24/45^†	100^†/100/ND/100/96/ND^†	100^†/69/ND/63/94/ND^†	0^†/0*/ND/3/6/10^†
8	57^†/22/34/18/45^†	100^†/100/100/100/ND^†	100^†/78/100/55/85^†	0^†/0/0/0/12^†
9	52^†/19/28/87	100^†/100/100/100^§	100^†/100/100/14	0^†/0/0/0
10	19	ND	ND	ND

ND, not done.
The numbers in black (roman font; not bold, italic or underlined entries) indicate low-density split, ∼4,000 cells per cm².
Bold entries indicate high-density split, ∼8,000–20,000 cells per cm².
Italic entries denote cells with extended lifespan on initial cultivation in BBMYAB and mouse feeders.
The entries that are underlined denote hTERT/shp16-transduced cells.
The percentage of ER^pos and lineage marker-positive cells evaluated after immunoperoxidase staining of cultures and counterstained with haematoxylin. Aside from staining for ER (SP1, prediluted), the lineage markers included luminal keratin K8 (stained by M20 or TS1) and keratin K19, and myoepithelial P63. Each number refers to the frequency of stained cells among a total of 1,000 cells in randomly selected fields in a culture representing one biopsy. Cells were counted by use of a × 25 objective and a × 10 ocular equipped with a grid. Note that cultures remain essentially ER^pos and luminal like. At the onset of senescence (starting at passage 4 in low-density split cultures and up to passage 6–9 in high-density split cultures) there is a tendency for the ER staining to fade out. High-density cultures from two biopsies maintained a high ER expression on omission of EGF and addition of E2.
^*Very few, small foci or individual cells.
^†EGF omitted, E2 added.
^‡+E2 in passage 1.
^§Stained in passage 11.

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