Establishment of Human Adipose Tissue-Derived Stromal Cell Lines: A Culture System to Manufacture Megakaryocytes Releasing Functional Platelets
Authors: Tozawa Keiichi , Ono-Uruga Yukako , Takizawa Noriko , Horiuchi Tadashi , Okamoto Shin-ichiro , Murata Mitsuru , Ikeda Yasuo , Matsubara Yumiko 
Published: October 2019

Abstract
We have described the use of adipose tissue-derived stromal (ADS) cells (also known as pre-adipocytes) to generate megakaryocytes (MKs) and platelets (Plts) for potential application as non-donor-derived Plts in transfusion medicine. ADS cells are an attractive candidate cell source, because (1) their differentiation does not require gene transfer as they utilize endogenous genes in differentiation into functional MKs, (2) megakaryopoiesis and thrombopoiesis are dependent on endogenous thrombopoietin, and (3) ADS cells are genetically stable in long-term culture. However, Plt yield remains insufficient for clinical application. In the present study, we focused on the establishment and characterization of cell lines from small amount of human ADS cells (5 x 105 cells isolated from ~0.5 g fat). To clarify the usefulness of ADS cell line (ASCL) as a source for clinical application, characterization of ASCL and platelet function tests were performed on ASCL-derived MKs and Plts. We modified the established method, utilizing an upside-down culture flask method (Yagi et al, BBRC 2004), to obtain fat cell line with multipotent capacity. Instead of using large volume of fat as described in the original method, we developed ASCL from a small number of ADS cells, because ADS cells possess the capacity of expansion and differentiation into mature adipocytes rapidly. These ASCL have now been expanded in culture at least 6 months. Gene expression analysis by quantitative real-time PCR indicated that ASCL expressed the genes for several pluripotent cell-markers, such as, OCT3/4, KLF4, Myc, Nanog, Gal, and GABRB3. Also, ASCL expressed genes related to MK lineages, such as p45NF-E2, RUNX1, GATA2, Fli1, FOG1, TPO, and c-MPL. These phenotypes did not differ from ADS cells. These cells were then cultured in MK induction media consisting of IMDM containing transferrin. The frequency of CD41+/CD42b+ MK-sized cells on day 10 was 24 ± 2%. DNA polyploidy, a hallmark of MKs, in CD41+ cells ranged from 2N to 16N with an average of 4.3N. These results were similar to MKs derived directly from ADS cells. The frequency of CD41+/CD42b+ Plt-sized particles on day 18 was 34 ± 6%. For Plt yield, ~30 Plts were obtained from single ASCL. Analysis of granule contents showed that ASCL-derived Plts contain ATP and ADP, and von Willebrand factor. We next examined functionality of these ASCL-derived Plts. Donor Plts were used as a control. Upon stimulation with thrombin (0.5 U/mL), binding of labeled fibrinogen occurred to both ASCL-derived Plts and donor Plts. But binding to ASCL-derived Plts was higher (p<0.001). We also tested PAC1 binding, a marker for Plt activation. Similar to fibrinogen binding, binding to ASCL-derived Plts was higher than to donor Plts (p<0.001). Also, ASCL-derived Plts spread on fibrinogen-coated glass in the presence of ADP/PAR1 (10 μM each), forming lamellipodia stained with anti-beta tubulin antibody and phalloidin. Clot retraction was observed upon stimulation (0.5 U/mL thrombin, 1.5 mM calcium, 100 μg/mL fibrinogen) of both ASCL-derived Plts and donor Plts. Sufficient number of Plts were obtained to perform Plt aggregation study under light transmission aggregometry. Agonist (ADP 20 μM, calcium 1.5 mM)-induced Plt aggregation was observed with ASCL-derived Plts, but not ASCL-derived CD41- particles. Taken together, we established human ADS cell line, ASCL. They retain the ability to proliferate and differentiate into Plts for at least 6 months, and therefore, may be suitable for manufacturing functional Plts by simple method.

Disclosures
No relevant conflicts of interest to declare.