Reversible Growth-Arrest of a Spontaneously-Derived Human MSC-Like Cell Line

Abstract

<jats:p>Life cycle limitation hampers the production of high amounts of primary human mesenchymal stroma-/stem-like cells (MSC) and limits cell source reproducibility for clinical applications. The characterization of permanently growing MSC544 revealed some differentiation capacity and the simultaneous presence of known MSC markers CD73, CD90, and CD105 even after continuous long-term culture for more than one year and 32 passages. The expression of CD13, CD29, CD44, and CD166 were identified as further surface proteins, all of which were also simultaneously detectable in various other types of primary MSC populations derived from the umbilical cord, bone marrow, and placenta suggesting MSC-like properties in the cell line. Proliferating steady state MSC544 exhibited immune-modulatory activity similar to a subpopulation of long-term growth-inhibited MSC544 after 189d of continuous culture in confluency. This confluent connective cell layer with fibroblast-like morphology can spontaneously contract and the generated space is subsequently occupied by new cells with regained proliferative capacity. Accordingly, the confluent and senescence-associated beta-galactosidase-positive MSC544 culture with about 95% G0/G1 growth-arrest resumed re-entry into the proliferative cell cycle within 3d after sub-confluent culture. The MSC544 cells remained viable during confluency and throughout this transition which was accompanied by marked changes in the release of proteins. Thus, expression of proliferation-associated genes was down-modulated in confluent MSC544 and re-expressed following sub-confluent conditions whilst telomerase (hTERT) transcripts remained detectable at similar levels in both, confluent growth-arrested and proliferating MSC544. Together with the capability of connective cell layer formation for potential therapeutic approaches, MSC544 provide a long term reproducible human cell source with constant properties.</jats:p>

Anti-Tumor Effects of Exosomes Derived from Drug-Incubated Permanently Growing Human MSC

“…Previous work has demonstrated that MSC544 can undergo transient cell cycle arrest during confluency. These changes from a proliferative to a growth-inhibited and senescent-like state of MSC544 cultures display reversible functional alterations associated with marked differences in metabolic activities [ 5 ]. Accordingly, we were interested to test presumably altered exosome production during this phase of confluency-mediated growth arrest.…”

“…MSC544 cease to grow after contact inhibition when the culture reaches confluency and develops a tissue-like structure [ 5 ]. Thereby, the cells enter a transient G0’-like arrest cycle, as previously suggested for a retrodifferentiation program and the role of protein kinase C signaling [ 54 , 55 , 56 ].…”

“…The transient G0’-like cell cycle arrest can be maintained until the culture is placed again in a subconfluent environment. Thereby, MSC544 re-enter the proliferative cell cycle paralleled by immediate down-modulation of senescence-associated markers [ 5 ]. During such reversible growth arrest, however, a markedly reduced exosome production was detectable, suggesting that release of MSC544-generated exosomes is predominantly associated with progressive cell growth.…”

“…The human MSC544 cell line reflects a permanently growing MSC population as extensively characterized in previous work [ 5 , 61 ]. MSC544 were cultured in an MSC growth medium (αMEM (Sigma Chemie GmbH, Taufkirchen, Germany) supplemented with 10% allogeneic human AB-serum (blood from 31 male AB donors was commercially obtained from a blood bank at Hannover Medical School, Germany, and processed to a serum), 100 U/mL penicillin, 100 µg/mL streptomycin, and 2 mM L-glutamine (Sigma Chemie GmbH) at 37 °C with 5% CO 2 in a humidified atmosphere.…”

“…This requires expansion of frequently new MSC populations and complicates reliability and reproducibility of the stem cell source, e.g., by donor heterogeneity. Thus, standardization with a permanently proliferating human MSC cell line model, such as MSC544, provides a unique cell source and reduces heterogeneity for potential clinical utilization [ 5 ].…”