Factors that govern LTCIC maintenance in stromal cultures are unknown. We previously showed that direct adhesive interactions between LTCIC and stromal ECM inhibit LTCIC-proliferation, However, coculture of LTCIC in contact with the murine fetal liver cell line AFT024[AFT] results in a significantly better maintenance of human LTCIC, than when cultured in transwells above the AFTfeeder. Furthermore, maintenance of LTCIC in contact with AFT is 3fold better than in contact with primary stroma or with the murine BM stromal cell line M2IOB4. The aim of the present study was to determine if the contact-inhibition previously described by our group does not occur in AFT cultures and/or to identity signal(s) produced by AFT cells that allows increased LTCIC-maintenance. Human 34+/DR- cells were cultured in artificial stroma conditioned medium (aSCM =IMDM, serum, N-desulfated heparin, pg cone, cytokines) in contact with glutaraldehyde fixed AFT, AFT-ECM or BSA. The LTCIC recovery after 5 weeks was significantly lower in fixed-AFT (13%±3) or AFT-ECM (!7%±2) cultures compared to BSA. This confirms that contact with ECM inhibits LTCIC proliferation and suggests that increased LTCIC maintenance in contact with AFT may not be due to ECM component(s) nor to cell surface expressed mofecules. Alternatively, continued production of factors) throughout the culture may be required to support LTCIC. We then added aSCM combined with IL-3 and MIPloc to cultures in wich 34+/DR- cells were cultured in contact with AFT-ECM, fixedAFT or BSA. After 5 weeks 89±12% of day 0 LTCIC were maintained in BSAcoated wells, consistent with the results we recently described for non-contact cultures with primary stroma. However, significantly less LTCIC were maintained when cocultured in aSCM+IL3+MIPla on AFT-ECM (49%±10) or fixed-AFT (40%±7). Interestingly when 34+/DR- cells were cultured in aSCM+IL-3+MlPla in contact with viable AFT, the number of LTCIC recovered after 5 weeks exceeded that of the input number significantly (149±I7%). Addition of IL-3+- MlPlct to cocultures with other viable stromal feeders such as M2-10B4 did not result in maintenance of LTCIC at week5 (36+4% maintenance). We describe here for the first time that LTCIC can be expanded for a minimum of 5 weeks ex vivo, using an AFT-contact culture system, aSCM , IL3 and MIP-loc. We demonstrate also that AFT024 cells produce one or more factor(s) that counteract contact-mediated proliferation inhibition. The nature of this factors) is unknown. We have recently shown that a cDNA identical with the transmembrane protein DLK is expressed in AFT024 cells and may play a role in the maintenance of murine stem cells on this feeder. We are currently investigating the role of DLK in the support of human LTCIC.
|Number of pages||1|
|State||Published - 1997|