Characterization of the interactions between stromal and haematopoietic progenitor cells in expansion cell culture models N.M. Bilko * , I.A. Votyakova, S.V. Vasylovska, D.I. Bilko Medical Centre of Tissue and Cell Therapy ‘‘Embryotech’’, National University ‘‘Kyiv-Mohyla’’ Academy, Kiev, Ukraine Received 31 August 2004; revised 2 November 2004; accepted 11 November 2004 Abstract Development of the long-term culture models of haematopoietic stem cells (HSCs) is one of the important tasks in modern biotechnology. It has been suggested that stromal presence is important for haematopoiesis in vitro and in vivo, but the question remains: whether diffusible factors produced by stromal cells are sufficient for the regeneration of primitive and definitive haematopoietic cells, or direct cell-to-cell contacts of the cultured material with underlying stromal base would be required. During present studies, influence of various feeder layers and feeder layer conditioned media on proliferative, differentiative and clonogenic activity of human AC133C derived from human umbilical cord blood was investigated. Cell extracts for feeder layers were prepared from 4e6 weeks old human embryos and co-cultured feeder cells. Effects of the conditioned media were also determined. Culture and feeder layer media were additionally supplemented with commonly implemented factors such as GM-CSF, IL-3 and LIF. Estimation of morpho-functional properties of AC133C cultivated suspension cultures was performed in subculture experiments using semisolid agar culture conditions. Multipotential CFU-MIX (CFU-GEMM) and unipotential progenitor cells CFU-GM, BFU-E and CFU-E were observed and analyzed. Our data suggest that haematopoiesis can be sustained for prolonged cultivation periods in the presence of feeder layer cells or conditioned media supported culture models. Prolonged support of primitive haematopoietic cells and their clonogenic capacity and functional characteristics in feeder layer positive cultures, indicates that diffusible factors are sufficient for haematopoiesis and suggests that direct cell-to-cell contacts may not be exclusively required for successful long-term in vitro haematopoiesis. Ó 2004 International Federation for Cell Biology. Published by Elsevier Ltd. All rights reserved. Keywords: Haematopoietic progenitor cells; Stromal cells; Growth promoting cytokines; Ex vivo cultural system 1. Introduction Application of the cell therapy methods into the day- to-day medical routine largely depends on the advances in cell biotechnology. One of the most important milestones in the cellular biotechnological methods would be determination of the factors that would allow increase of the functional progenitors that can be implemented in therapeutic purposes (Bilko, Culture of erythroid precursor cells, 1997; Butenko, 1978; Vladimirskaja and Rymjantsev, 2000). In contrast to other in vitro cell models that are characterized by straight forward proliferation of the cultured cells and formation of the identical populations, haematopoietic system has finite and limited capacity for proliferation in vitro, and terminally differentiates leading to the inevitable demise of the population (Askenasy et al., 2003; Chertkov et al., 1990; Maitra et al., 2004). Information about complexity of the factors required for prolonged artificial proliferation, and pluripotency of the haematopoietic stem cells are opening new pos- sibilities for the research in this field of science (Goodell et al., 2001; Wobus, 2001). Interactions of the stromal component with haematopoietic cells are of a great * Corresponding author. E-mail addresses: nadja@bilko.kiev.ua, info@embryotech.com.ua (N.M. Bilko). 1065-6995/$ - see front matter Ó 2004 International Federation for Cell Biology. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.cellbi.2004.11.016 www.elsevier.com/locate/cellbi Cell Biology International 29 (2005) 83e86