Expansion of Murine Amniotic Fluid c-Kit High Lin- Cells Maintaining Their Hematopoietic Potential.

Abstract 1475

Poster Board I-498

We have recently described the hematopoietic potential of ckit+ Lin- cells from the murine and human amniotic fluid (1). These cells were able to generate all types of blood cells in vitro and as far as mice are concerned, to generate a complete hematopoietic system once transplanted to immunodeficient recipients. This strong hematopoietic potential was accompanied by a molecular signature measured by unicellular RT-PCR, characteristic of fetal hematopoietic progenitors. Indeed coexpression of Gata2, Lmo2 and Aml1 was found in 28% of ckit+Lin- AF cells.

Intriguingly, murine ckit+Lin- AF cells can be subdivided into two fractions depending on the level of ckit expression (low or high). In in vitro assays, we demonstrated that hematopoietic potential was strictly restricted to the ckit high expressing fraction. The expansion of these cells would have great impact even in the clinical field as AF could be seen as a source of transplantable hematopoietic stem cells (HSCs). Many of the early studies that documented some expansion ability of HSCs included fetal calf serum in the protocol. Given the poorly defined combination of factors in serum and the variability between different serum lots, these protocols were often difficult to reproduce. Serum-free media supplied with specific inducers have been shown to bring several advances in driving direct differentiation of embryonic stem cells. Murine AF Lin-ckit^lo and ckit^hi were cultured in serum- and feeder layer-free culture conditions. Lin-ckitlo AF cells died within a few days. Conversely, Lin-ckithi AF cells were maintained for up to 6 weeks, with a proliferation rate of more than 100 during the first three weeks. Their phenotype remained stable, ckit^hi CD45+ and Lin-. The hematopoietic potential tested in methylcellulose assays showed an increased frequency of mixed CFU-GEMM (from 24% to 84%). In vivo, CFU-S₁₂ composed of erythroid, myeloid and Lin-ckit+Sca1+ progenitor cells were observed after the injection of AF ckit^hi in lethally irradiated recipients. Gene expression profile analyzed by single cell multiplex RT-PCR analysis correlated with the in vitro and in vivo results of differentiation. LMO2 was coexpressed with Gata2 and Aml1 in 66% of expanded cells, demonstrating the maintenance of an overall pattern of expression.

Collectively, our results indicate that the hematopoietic potential of AF resides in the ckit^hi fraction and that these cells can be expanded in serum-free condition for prolonged periods of time without reduction of their hematopoietic potential. This strongly supports the idea that AF may be an excellent source of cells for therapeutic applications.

1. Ditadi 2009