Glycogen synthase kinase-3b (GSK-3b) has been identified as an important regulator of stem cell function acting through activation of the wingless (Wnt) pathway. Here, we report that treatment with an inhibitor of GSK-3b, 6-bromoindirubin 3 0 -oxime (BIO) delayed cell cycle progression by increasing cell cycle time. BIO treatment resulted in the accumulation of late dividing cells enriched with primitive progenitor cells retaining the ability for sustained proliferation. In vivo analysis using a Non-obese diabetic/ severe combined immunodeficient (NOD/SCID) transplantation model has demonstrated that pretreatment with BIO promotes engraftment of ex vivo-expanded hematopoietic stem cells. BIO enhanced the engraftment of myeloid, lymphoid and primitive stem cell compartments. Limiting dilution analysis of SCID repopulating cells (SRC) revealed that BIO treatment increased human chimerism without increasing SRC frequency. Clonogenic analysis of human cells derived from the bone marrow of transplant recipient mice demonstrated that a higher level of human chimerism and cellularity was related to increased regeneration per SRC unit. Gene expression analysis showed that treatment with BIO did not modulate the expression of canonical Wnt target genes upregulated during cytokine-induced cell proliferation. BIO increased the expression of several genes regulating Notch and Tie2 signaling downregulated during ex vivo expansion, suggesting a role in improving stem cell engraftment. In addition, treatment with BIO upregulated CDK inhibitor p57 and downregulated cyclin D1, providing a possible mechanism for the delay seen in cell cycle progression. We conclude that transient, pharmacologic inhibition of GSK-3b provides a novel approach to improve engraftment of expanded HSC after stem cell transplantation.

We propose a quantitative method to characterize growth and differentiation dynamics of multipotent cells from time series carboxyfluorescein diacetate, succinimidyl ester (CFDA-SE) division tracking data. The dynamics of cell proliferation and differentiation was measured by combining (CFDA-SE) division tracking with phenotypic analysis. We define division tracking population statistics such as precursor cell frequency, generation time and renewal rate that characterize growth of various phenotypes in a heterogeneous culture system. This method is illustrated by study of the divisional recruitment of cord blood CD34 1 cells by hematopoietic growth factors. The technical issue of assigning the correct generation number to cells was addressed by employing high-resolution division tracking methodology and daily histogram analysis. We also quantified division-tracking artifacts such as CFDA-SE degeneration and cellular autofluorescence. Mitotic activation of cord blood CD34 1 cells by cytokines commenced after 2 days of cytokine stimulation. Mean generation number increased linearly thereafter, and it was conclusively shown that CD34 1 cells cycle slower than CD34 2 cells. Generation times for CD34 1 and CD34 2 cells were 24.7 6 0.8 h and 15.1 6 0.9 h (6SD, n 5 5), respectively. The 20-fold increase in CD341 cell numbers at Day 6 could be attributed to a high CD34 1 cell renewal rate (91% 6 2% per division). Although cultures were initiated with highly purified CD34 1 cells ($96%), CD34 2 numbers had expanded rapidly by Day 6. This rapid expansion could be explained by their short generation time as well as a small fraction of CD34 1 cells ($5%) that differentiated into CD34 2 cells. Multitype division tracking provides a detailed analysis of multipotent cell differentiation dynamics. ' International Society for Analytical CytologyKey terms cell division tracking; cord blood stem cells; in vitro expansion; carboxyfluorescein diacetate succinimidyl ester LYONS and Parish were the first investigators to report serial halving of fluorescence intensity of lymphocytes stained with the vital dye CFDA-SE (carboxyfluorescein diacetate, succinimidyl ester) (1). The technique provided an important adjunct to cell cycle analysis by DNA measurement. Flow cytometry was used to identify and gate as many as eight consecutive cell cycles and so analyze the changes in expression of internal or external molecules with respect to cell generation number. The technique was originally applied to the study of lymphocyte division because of their homogeneous staining properties. By employing a simple sorting strategy, Nordon et al. (2) demonstrated that it was possible to obtain a similarly well-defined cluster pattern with other cell types that did not stain homogeneously. This technique has had an important role to play in the study hematopoietic stem cell quiescence, renewal, and differentiation (3-10).To date, the assignment of cell generation number to CFDA-SE clusters has been an empiric process that is heavily reliant o...

No abstract

No abstract