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Cell cycle and functional differences between CD34+/CD38hi and CD34+/38lo human marrow cells after in vitro cytokine
1Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, WA 98104.
Insights
Human marrow cells with CD34+/38lo (CD38lo) phenotype exhibit distinct proliferation kinetics and clonogenic potential compared to CD34+/38hi (CD38hi) cells. CD38lo cells maintain their phenotype and a proportion of clonogenic capacity after in vitro expansion with growth factors.
Area of Science:
- Hematology
- Cell Biology
- Stem Cell Research
Background:
- Hematopoietic stem cells (HSCs) are crucial for blood cell production.
- Cell surface markers like CD34 and CD38 help differentiate HSC subpopulations.
- Understanding the behavior of these subpopulations under culture conditions is vital for regenerative medicine and research.
Purpose of the Study:
- To compare the proliferation and clonogenic activity of CD34+/38hi (CD38hi) and CD34+/38lo (CD38lo) human marrow cells.
- To investigate the effects of in vitro culture with recombinant growth factors on these cell populations.
- To assess the long-term clonogenic potential of CD38lo cells after expansion.
Main Methods:
- Human marrow cells were isolated and categorized as CD38hi or CD38lo.
- Cells were cultured in serum-deprived medium with a cocktail of recombinant growth factors (IL-1, IL-3, IL-6, G-CSF, GM-CSF, kit ligand, erythropoietin).
- Proliferation kinetics (S-phase entry) and clonogenic activity (colony-forming units, CFU) were measured before and after 6 days of culture. Phenotypic analysis was performed at day 6.
Main Results:
- Before culture, CD38hi cells showed higher S-phase fraction (13%) than CD38lo cells (3%).
- CD38hi cells had greater short-term CFU, but CD38lo cells possessed significantly higher long-term pre-CFU potential (107 colonies/1,000 cells vs. 3 colonies/1,000 cells).
- After 6 days of culture, CD38hi cells showed a higher proliferative response (44.2% cycling at 24h) than CD38lo cells (5.1% cycling at 24h, rising to 28% at 72h).
- At day 6, 86% of CD38hi cells lost the CD34+/38hi phenotype, while 60% of CD38lo cells retained the CD34+/38lo phenotype.
- In vitro-expanded CD38lo cells showed a twofold decrease in clonogenic activity, with loss of erythroid and GM colony potential, but a proportion capable of generating CFU was maintained.
Conclusions:
- CD34+/38lo cells represent a distinct subpopulation with higher long-term stem cell potential compared to CD34+/38hi cells.
- In vitro expansion with growth factors differentially affects these subpopulations, with CD38hi cells undergoing more rapid proliferation and phenotype changes.
- A subset of CD38lo cells retains clonogenic capacity after in vitro expansion, suggesting their potential utility in cell-based therapies, although their differentiation potential may be altered.
Abstract:
The proliferation kinetics and clonogenic activity of CD34+/38hi (CD38hi) and CD34+/38lo (CD38lo) human marrow cells were measured before and after culturing the cells in vitro over a 6-day period in serum-deprived medium containing recombinant growth factors (interleukin-1 [IL-1], IL-3, IL-6, granulocyte colony-stimulating factor [G-CSF], granulocyte-macrophage [GM]-CSF, kit ligand, and erythropoietin). Before in vitro culture, 3% +/- 3% of the CD38lo and 13% +/- 2% of the CD38hi cells were in the S-phase of the cell cycle. The clonogenic activity of CD38hi cells was twofold greater than that of the CD38lo cells, as measured by colony-forming units (CFU) in short-term assays. However, CD38hi cells contained fewer pre-CFU than did the CD38lo cells, generating only 3 +/- 2 colonies per 1,000 cells after 4 weeks of culture on competent stromal layers, compared with 107 +/- 46 colonies per 1,000 cells from the CD38lo population. CD38hi and CD38lo cells exhibited distinctly different responses when cultured in serum-deprived medium supplemented with recombinant growth factors. After culturing cells for 24 hours, CD38lo cells essentially remained a noncycling population with only 5.1% +/- 3.0% of the cells cycling, whereas 44.2% +/- 6.9% of the CD38hi cells were in DNA synthesis. Gradually CD38lo cells were recruited into cycle, such that by 72 hours, approximately 28% of the CD38lo cells were in S-phase. However, during 6 days of culture, the percentage of cycling CD38lo cells never exceeded the proliferative response observed for CD38hi cells. Phenotype analysis conducted at day 6 indicated that 86% of the CD38hi population were no longer phenotypically CD34+/38hi, while 60% of CD38lo cells maintained a CD34+/38lo phenotype. Long-term cultures initiated with 6-day in vitro-expanded CD38lo cells showed approximately a twofold decrease in clonogenic activity attributable to a loss of erythroid precursors and a decrease in GM colonies. Thus, a proportion of CD38lo cells capable of generating CFU was maintained even after exposure to growth factors.