When seeded at clonal density, CD34+ keratinocytes formed larger colonies than CD34– cells (p<0.05), indicating a higher proliferative potential.
In this new culture system, these human pluripotent stem cells can stably grow, even if in clonal density with keeping expression of stem cell markers.
When plated above clonal density (10 cells/µl) many spheres formed by 1 week (Fig. 1A).
The increased CFE when cells are plated at a clonal density is intriguing and further studies are needed to prove that the colonies formed at clonal density are also an indication that some of these are stem cells.
Complete Tcf3 deficiency greatly reduced differentiation and allowed ES cell expansion without exogenous LIF, even at clonal density.
Next we determined whether we could obtain stable cell lines from STAT5bERpos memory B cells isolated from clonal density cultures.
Half of the lines readily formed tumorspheres when plated at clonal density in serum-free neural stem cell conditions.
SP and MP cells were separately seeded at a clonal density of 400 cells/cm2 in 35 mm dishes and cultured in EGM-2MV medium (Cambrex, Walkersville, MD).
We used these genes as markers of regional identity and found that their differential expression was maintained in vitro after multiple passages at clonal density (Figure 3).
The self-renewing capacity of these tumorspheres was assessed by dissociating them into single cell and growing at a clonal density of 1,000 cells per milliliter.
To evaluate the self-renewal capability, the formed spheroids were disintegrated into single cells and further cultured at a clonal density in hESCM.
