The majority of available human stable cell lines have cancer or embryonic origin or their immortalizations were achieved by introduction of vectors with some genes. Hence, there is a demand for new human cell lines, especially obtained from normal tissues of adult organism.
In the department of human genetics of Institute of molecular biology and genetics of NAS of Ukraine the new human stem cell line 4BL was obtained from peripheral blood of adult healthy donor, using feeder and special medium with recombinant cytokins LIF, SCF and IL-3 (Lukash 2011). Karyotype evolution of such cell lines is virtually not investigated. Taking into account that cell lines are valuable objects for various research, regular monitoring of their genome changes is essential for proper data interpretation.
Cell line 4BL had successfully overcome the Hayflick limit and is cultivated for more than 220 passages (above 10 years) in culture. For the first time, its karyotype analyses were performed at different stages of establishment and stabilization. The comparison with stem and cancer cell line was done and the following regularities were revealed: sex chromosome loss during long-term cultivation and non-random character of chromosome aberrations – which is close to changes in organism with age or disease. We detected a polyploidization of the part of cell population at late passages (196th -221st passages), which is a characteristic of rodent cell lines or some cancer cell lines. The hallmark of the cell line 4BL is six marker chromosomes and the unique complex of chromosomal aberrations: t(1;11)(q12;p15), der(2)(p11~12), t(5;15)(q10;p10), t(12;15)(p10;q10), t(16;21)(q13;p11), despite which the near-diploid modal class in 42-43 chromosome is maintained for more than 10 years in culture.
FISH Cytocell OctoChrom technique application allowed not only to clarify the nature of chromosomal aberrations, detected by G-banding, but also to identify some marker chromosome, which appeared to be derivatives of 4 and 17 chromosome.
We have revealed by array CGH that majority of chromosomal aberrations are mosaic, what confirmed the presence of different clones in the cell population of line 4BL. The feature of karyotype stabilization stage can be considered main chromosomal rearrangements (duplications of chromosomes 2, 10, 16 and 19 and deletions of chromosomes 4, 10, 12, 13, 17 and Х), which are maintained in cells from 120th till 205th passage, whereas small new aberrations were eliminated on the next investigated passage. The selective advantages of the continuously reproduced chromosome aberrations can be associated with the increased genes dose of mitotic signal pathways (dup19р13.3-р12) on the background of the decreased copy number of the tumor suppressor genes (del 13q12.11-q21.2). As a result the enhanced proliferative capacity is accompanied by increased gene dosage of apoptosis inducers (dup2q31.1-q33.1, dup16q12.1q12.2, dup16 q22.1-q24.3).
It has been shown that addition of stress factor into the system (high ionic strength of medium) changed the stabilizing selection to the directional one and provoked a new wave of genomic instability growing, what is the feature of establishment stage, and had a positive correlation with MGMT expression.
For the first time we investigated morphological and growth peculiarities of new cell line 4BL. Presence of two cell types – fibroblast-like and epithelioid-like – was revealed. The abilities of these cells to form rounded colonies in soft agar and to differentiate into adipogenic, myogenic and osteogenic directions were determined.
By flow-cytometry results, cell populations of line 4BL are positive for markers of mesenchymal stem cells CD73+ and CD105+, and are negative for hematopoietic stem cell markers CD14-, CD34- and CD45-, also cells are negative for Oct4-, which is typical
for the cells of embryonic or tumor origin. So, cell populations of line 4BL most probably belong to multipotent non-hemopoietic stem cells.
The immortalization of cell line 4BL has been confirmed by telomerase expression at different passages. Cell line 4BL can be recommended as a model object for investigation of fundamental processes: adaptation to long-term cultivation in vitro, karyotype evolution under regressive condition, and, considering its more than 10 years maintenance in culture – as a model of human cell changes during aging. It is possible to use this cell line for drugs testing and for development of skin equivalents. Taking into account the origin of the 4BL line – peripheral blood of adult healthy donor – it can be used as a control for research on cancer or artificially immortalized cell lines.
