ROCK Inhibitor and Feeder Cells Induce the Conditional Reprogramming of Epithelial Cells

Abstract

We demonstrate that a Rho kinase inhibitor (Y-27632), in combination with fibroblast feeder cells, induces normal and tumor epithelial cells from many tissues to proliferate indefinitely in vitro, without transduction of exogenous viral or cellular genes. Primary prostate and mammary cells, for example, are reprogrammed toward a basaloid, stem-like phenotype and form well-organized prostaspheres and mammospheres in Matrigel. However, in contrast to the selection of rare stem-like cells, the described growth conditions can generate 2 × 10(6) cells in 5 to 6 days from needle biopsies, and can generate cultures from cryopreserved tissue and from fewer than four viable cells. Continued cell proliferation is dependent on both feeder cells and Y-27632, and the conditionally reprogrammed cells (CRCs) retain a normal karyotype and remain nontumorigenic. This technique also efficiently establishes cell cultures from human and rodent tumors. For example, CRCs established from human prostate adenocarcinoma displayed instability of chromosome 13, proliferated abnormally in Matrigel, and formed tumors in mice with severe combined immunodeficiency. The ability to rapidly generate many tumor cells from small biopsy specimens and frozen tissue provides significant opportunities for cell-based diagnostics and therapeutics (including chemosensitivity testing) and greatly expands the value of biobanking. In addition, the CRC method allows for the genetic manipulation of epithelial cells ex vivo and their subsequent evaluation in vivo in the same host.

Efficient ultrafiltration‐based protocol to deplete extracellular vesicles from fetal bovine serum

“…Metabolic activity and total DNA count for each cell type are shown in the Supplement Figure 2. In addition, we tested UF-dFBS for culturing of mouse 3T3 cells and conditionally reprogrammed cells [26] derived from prostate (as described in [19])  and renal cancer [Saeed et al, unpublished results] patient tissue samples. All cell types cultured for up to 72h ( n  = 12) exhibited no apparent arrest in the proliferation (data not shown).…”

Expansion of airway basal epithelial cells from primary human non‐small cell lung cancer tumors

“…Traditionally, the primary culture of human cancer cells has been challenging, with few tumors amenable to culture on plastic, so this protocol, known as “conditional reprogramming” or “3T3 + Y,” has naturally attracted attention in the cancer community. To date, variants of this protocol have allowed cancer cell cultures to be established across multiple cancer types including lung, prostate, pancreas and colon 4, 5, 6…”

Telomerase enzymatic component hTERT shortens long telomeres in human cells

“…15 This culture condition dramatically upregulates endogenous hTERT expression level (8-to 14-fold increase) and telomerase activity. 15 Under the feeder layer co-culture condition, GUMC001 cells (human primary prostate epithelia cells) maintained relatively small and constant TLV and no significant change in average telomere length per telomere from PD6 to PD48, whereas the number of excessively long telomeres remained stably low, and the number of very short telomeres decreased from PD6 to PD48 ( Table 2). The observed small TLV and low number of excessively long and very short telomeres in GUMC001 cells are comparable with what was observed in hTERT-expressing IMR90 cells ( Table 1).…”

“…14 Briefly, chromosome preparations were dropped onto clean microscopic slides and hybridized with 15 μl of hybridization mixture consisting of 0.3 μg/ml Cy3-labeled telomere-specific peptide nucleic acid (PNA) probe, 1 μl of cocktails of FITC-labeled centromeric PNA probes specific for chromosomes 2,4,8,9,13,15,18,20, and 21, and 20 μg/ml of Cy3-labeled centromeric PNA probes specific for chromosome X (Biomarkers), in hybridization buffer containing 50% formamide, 10 mM TRIS-HCl, pH 7.5, and 5% blocking reagent. Slides were denatured and then hybridized at 30 °C for 3 h. After hybridization, the slides were sequentially washed 10 min each at 42 °C: once in 1 × SSC, once in 0.5 × SSC, and once in 0.1 × SSC.…”