Assessment of Differentiation States of Hematopoietic Stem Cells Following in Vitro Culture Using Side and Forward Scatter of Flow Cytometry

Yongxin Zhang, Farhang Farhangfar, Leonard Giblin, David Wolf, Francesca Vitelli, Yu Zhou, Donnie Rudd

Hematopoietic stem cells (HSC) are defined by the International Society of Hematotherapy and Graft Engineering (ISHAGE) as those at low side scatter, positive for CD34 and CD45dim for their numeration with flow cytometry. However, we found that these CD34+ cells increase their granularity and size following in vitro culture, which was exhibited in flow cytometry as more events at higher side scatter and forward scatter. To further determine whether such a change in the cell event distribution is related to HSC differentiation, HSC markers and differentiation markers of in vitro-cultured HSC were detected by flow cytometry at different side scatter and forward scatter levels using modified ISHAGE gating strategies. The results revealed that cultured HSC with higher side scatter have a lower percentage of cells positive for HSC markers and a higher percentage of differentiation makers, while those with higher forward scatter have a higher percentage of differentiation makers but a slightly higher percentage of stem cell markers, suggesting that side scatter and forward scatter levels of cultured HSC correlate with the differentiation level of these cells.