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Study of lymphocyte development from embryonic stem cells in vitro

University of British Columbia

Embryonic stem (ES) cells have been shown to differentiate to all hematopoietic lineages. However, lymphocyte development from murine ES cells has not been fully investigated. In this thesis, the developmental pathway of natural killer (NK) lymphocytes from ES cells was investigated, using a multi-step in vitro ES cell differentiation system. ES cells were induced to differentiate into embryoid bodies (EBs). CD34⁺ EB cells were isolated and cultured on OP9 stroma with a cocktail of cytokines to generate cells; termed ES-derived hematopoietic progenitors (ES-HPs). EB cell subsets as well as ES-HPs were tested for NK, T, B, and myeloid lineage potentials using lineage specific differentiation cultures. ES-HPs derived from CD34⁺ EB cells appeared to be heterogeneous and contained NK, T, B and myeloid potentials. At the EB level, lymphoid potential was found in CD34⁺CD45⁻ EB cell subset, while CD34⁺CD45⁺ EB cells had only myeloid but not lymphoid potential. CD34⁺CD45⁻ EB cells gave rise to CD45⁺ Mac-1⁻Ter119⁻(Lin⁻) ES-HPs, which were highly enriched for NK progenitors, but also had other lineage potentials. The NK progenitors among ES-HPs lacked CD122, a marker for NK lineage committed precursors, but they acquire CD122 as they differentiate along the NK lineage. To further enrich lymphoid progenitors among CD45⁻ EB cells, EB cell populations were dissected according to several surface markers and tested for myeloid and lymphoid lineage potentials. Hematopoietic progenitors with lymphoid potential in EBs were found to be CD45⁻CD34⁺c-kit⁺CD41⁺CD31⁺Flk-1⁺. As they differentiate in vitro into more mature hematopoietic progenitors, they slowly acquire CD45 and lose Flk-1 and CD41 expression while retaining erythroid/myeloid and lymphoid potentials. This study suggests that hematopoietic progenitors in EBs are similar to immature embryonic hematopoietic stem cells (HSCs) and they differentiate into more mature type HSCs in vitro.

Text

2011-02-10

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http://hdl.handle.net/2429/31170

Genetics

University of British Columbia

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