Cell Culture Systems Inspired by the Hematopoietic Stem Cell NichePublic Deposited
The hematopoietic stem cell (HSC) niche is the site where HSCs reside in vivo. The HSC niche is formed by supporting cells that provide structure and appropriate environmental cues for HSCs. In this study, three components of the HSC niche were evaluated for their influence on HSC expansion ex vivo: (1) culture in reduced oxygen tension, (2) presentation of TPO-mimetic (TPOm) lipid-peptide constructs in a hybrid-bilayer membrane, and (3) immobilization of stem cell factor (SCF) for cell culture. The HSC niche is localized to areas of reduced oxygen tension and others have shown that reduced pO2 cultures enhance the maintenance of HSCs. We hypothesized that a detailed analysis of HSC division would clarify how reduced oxygen tension alters HSC maintenance. However, using CFSE-labeled CD34+ cells, our results showed little difference in cell division profile, overall cellular expansion, or expansion/retention of CD34+Thy1+ cells between low oxygen tension and normoxia. Three forms of TPOm were synthesized: PEGylated TPOm (TPOm-PEG) and two forms of lipid-tethered TPOm (TPOm-L). Using M07e cells, we determined that TPOm-PEG and TPOm-L elicited similar activation of the signaling molecules ERK 1,2 and STAT5. Studies evaluating the expansion of HSCs showed similar levels of overall expansion and fraction of CD34+Thy1+ cells for TPOm-L and TPOm-PEG. Thus, TPOm-L did elicit cellular responses, but did not enhance expansion of HSCs as compared to soluble TPOm-PEG. In vivo, SCF is presented in both membrane-associated and soluble forms. Membrane-associated SCF has been shown to promote HSC self-renewal, while soluble SCF promotes the expansion of lineage-committed progenitors. Two different systems with biotin-binding molecules were used to evaluate our hypothesis that immobilization of SCF would improve HSC expansion. Investigation of the first system (commercially-available NeutrAvidin-coated plates) indicated that biotinylated-SCF released from the surface; therefore, the results could not be attributed directly to immobilized SCF. The second system incorporated a non-fouling, biotinylated poly(ethylene glycol) (PEG) molecules anchored to TiO2-coated surfaces via 2,4-dihydroxyphenylalanine (DOPA). Biotinylated-SCF was immobilized to the surface via an avidin bridge to biotin-DOPA-PEG. Surfaces characterized using M07e cells showed that immobilized biotinyated-SCF can elicit a specific adhesive interaction, activate ERK 1/2, and promote cell growth.
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