Megakaryocytes and Functional Platelets Obtained from Human Subcutaneous Adipocytes in an In Vitro Differentiation System.

Because of the difficulty in obtaining the sufficient amounts of hematopoietic stem cells from bone marrow, peripheral blood, and cord blood cells, studies of an in vitro experimental system that allows the production of abundant number of megakaryocytes (MKs) and platelets are currently the focus of research. Here, we present a novel system where human subcutaneous adipocytes were successfully differentiated into MK lineages in an in vitro liquid culture system. We also show that subcutaneous preadipocytes could be successfully transfected with vectors, to obtain modified MKs. Primary human subcutaneous preadipocytes (Cambrex Bio Science Walkersvile, Inc. Walkersville, MD, USA) were cultured in conditioned media to differentiate into mature adipocytes. Cells were cultured in serum-free media containing thrombopoietin for differentiation into MK lineages. The MKs or platelets were counted by flow cytometric analysis on day 14 using the relative value of CD41(+)/propidium iodide(+) cells or platelet size CD41(+) cells, respectively, versus 10⁷ subcutaneous preadipocytes on day 0. The MK and platelet cell count was approximately 9600/ 10⁷ and 2200/ 10⁷, respectively. Morphological analysis with electron microscopy demonstrated that MKs, which had typical organelles such as granules, demarcation membrane, and nuclei, and platelets, which had typical contents such as granules, mitochondria, and open canalicular system, were successfully obtained from subcutaneous preadipocytes. We then attempted to transfect subcutaneous preadipocytes with vectors to obtain transformed MKs. Two glycoprotein (GP) Ib alpha polymorphisms, ¹⁴⁵Thr/Met and 1–4 repeats of variable number tandem repeat of 13 amino-acid sequences, were used as the marker of gene transfer, which were detected by PCR-restricited fragment length polymorphism (RFLP) and Western blot analysis, respectively. Subcutaneous preadipocytes with the ¹⁴⁵Thr/Thr and 1 repeat sequences were transfected with the expression vector carrying GPIb alpha with the ¹⁴⁵Met and 4 repeats sequences. PCR-RFLP analysis with gel electrophoresis was performed on each RNA sample from the expression vector-transfected and non-transfected cells. Non-transfected cell sample had bands corresponding to the ¹⁴⁵Thr sequence position, while the expression vector-transfected cell sample had bands corresponding to both the ¹⁴⁵Thr and Met sequences. Western blot analysis with an anti-GPIb alpha monoclonal antibody, LJ-Ib alpha1 (a generous gift from Dr. ZM Ruggeri, The Scripps Research Institute, La Jolla, CA, USA), showed bands of the expected size corresponding to 1R and 1R/4R. In summary, we established an in vitro culture system to produce MKs and platelets from subcutaneous adipocytes. We were also able to obtain transfected MKs from subcutaneous preadipocytes.