(367j) Characterizing the Heterogeneity of Stem Cell Populations Useful for Transplantation

Stem cells are essential for cell replacement therapy because they differentiate into multiple distinct cell types, secrete bioactive molecules, and in some cases have positive immunomodulatory effects. Stem cell cultures are heterogeneous containing stem cells, partially differentiated progenitor cells, and fully differentiated cells, each with unique cell membrane features lending to their therapeutic diversity. However, stem cells’ natural heterogeneity presents limitations in transplantation therapies and hinders our understanding of their basic biological functions. Sufficiently characterizing stem cells’ functional behavior before using them in transplant therapy is essential to the development of reliable treatment options. In this work, two therapeutically relevant human stem cell populations, mesenchymal and neural, were screened using a multimodal profile consisting of cell size, cell proliferation, dielectrophoresis spectra, membrane capacitance, and cytoplasm conductivity. Additionally, differentiation gene expression and surface integrin expression were assessed for mesenchymal and neural stem cells, respectively. Using dielectrophoresis, a label-free cell analysis technique, we found that bone marrow-derived and adipose-derived mesenchymal stem cells have unique cytoplasm conductivity and similar membrane capacitance values. Through computer simulation, we identified that the transient slope of the dielectrophoresis spectra can be used as a metric to quantify the relative dielectric heterogeneity that exists within the human mesenchymal stem cell populations. Two sets of the neural stem cells derived from GMP-grade human embryonic stem cells, Shef4 and Shef6. With these cells, we generated three batches of Shef4 (4-1, 4-2, 4-3), a batch of Shef6 sorted (6S), and unsorted (6U) cells. We found that the 4-1, 4-2, and 4-3 cells have significant changes in size, proliferation, membrane capacitance, and integrin expression. The membrane capacitance depends on the passage number for the 4-1 and 4-2 cells (gradually decreasing). For alpha6 integrin, there was a strong correlation to the membrane capacitance of 4-2 cells (R²=0.882), and essentially no correlation to 4-3 cells (R²=0.081). For alphaV integrin, there was a weak correlation to the capacitance of 4-2 cells (R²=0.244), and no correlation for the 4-3 cells (R²=0.082). We assessed Shef6 cells because they were previously effective in a rodent spinal cord injury model. The Shef6 cells were FACS sorted for CD133+/CD34- markers producing the 6S batch of cells. The 6U batch of cells was not FACS sorted. Our results show that the 6S and 6U cells have significant changes in size, proliferation, membrane capacitance, and protein expression. The 6S cells were smaller and more proliferative than 6U cells. From the dielectrophoresis screening, 6S cells have higher membrane capacitance than 6U cells. Also, there was no difference in the expression of alphaV integrin for 6S and 6U cells. Significant variability exists among the bone marrow and adipose-derivedmesenchymal stem cells and the Shef4 and Shef6 cells. Dielectrophoresis provides a good quantitative assessment of cell phenotype variability. Careful screening to assess cellular heterogeneity will be critical for the development of robust stem cell therapies.