Characterization of Developmental Pathway of Natural Killer Cells from Embryonic Stem Cells In Vitro

In vitro differentiation of embryonic stem (ES) cells is often used to study hematopoiesis. However, the differentiation pathway of lymphocytes, in particular natural killer (NK) cells, from ES cells is still unclear. Here, we used a multi-step in vitro ES cell differentiation system to study lymphocyte development from ES cells, and to characterize NK developmental intermediates. We generated embryoid bodies (EBs) from ES cells, isolated CD34(+) EB cells and cultured them on OP9 stroma with a cocktail of cytokines to generate cells we termed ES-derived hematopoietic progenitors (ES-HPs). EB cell subsets, as well as ES-HPs derived from EBs, were tested for NK, T, B and myeloid lineage potentials using lineage specific cultures. ES-HPs derived from CD34(+) EBs differentiated into NK cells when cultured on OP9 stroma with IL-2 and IL-15, and into T cells on Delta-like 1-transduced OP9 (OP9-DL1) with IL-7 and Flt3-L. Among CD34(+) EB cells, NK and T cell potentials were detected in a CD45(−) subset, whereas CD45(+) EB cells had myeloid but not lymphoid potentials. Limiting dilution analysis of ES-HPs generated from CD34(+)CD45(−) EB cells showed that CD45(+)Mac-1(−)Ter119(−) ES-HPs are highly enriched for NK progenitors, but they also have T, B and myeloid potentials. We concluded that CD45(−)CD34(+) EB cells have lymphoid potential, and they differentiate into more mature CD45(+)Lin(−) hematopoietic progenitors that have lymphoid and myeloid potential. NK progenitors among ES-HPs are CD122(−) and they rapidly acquire CD122 as they differentiate along the NK lineage

Study of lymphocyte development from embryonic stem cells in vitro

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.Medicine, Faculty ofMedical Genetics, Department ofGraduat

T, B and myeloid potential of ES-HPs derived from CD34⁺CD45⁻ EB cells.

<p>(A) CD34⁺CD45⁻ EB cells were sorted and cultured with OP9 and cytokines to generate ES-HPs. They were then cultured on OP9-DL1 stroma with cytokines for T cell differentiation. At different time points, cells were harvested and stained for CD44 and CD25 and analyzed by flow cytometer. Dead cells were stained with propidium iodide and OP9-DL1 cells expressing green fluorescent protein were gated out. The numbers show the percentages of cells in the quadrants. (B) ES-HP cells were cultured for T cell differentiation for 8 days with 5 ng/ml IL-7 as in (A). On day 8, IL-7 concentration was reduced to 1 ng/ml and cells were cultured for an additional 6 days. Cells were harvested and analyzed by flow cytometer for CD4 and CD8 expression. The numbers indicate the percentages of cells in the quadrants. (C) ES-HP cells were cultured for T cell differentiation with 5 ng/ml IL-7 for 2 weeks and an additional 4 days with 1 ng/ml IL-7, stained for TCRγδ and TCRβ and analyzed by flow cytometer. The numbers show the percentages of cells positively stained with the test antibodies (filled histogram) over control antibody (open histogram). (D) ES-HPs were generated from CD34⁺CD45⁻ EB cells as in (A). The bulk and sorted CD45⁺Lin⁻ ES-HP cells were analyzed for T progenitor frequency by limiting dilution cultures as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000232#pone-0000232-g003" target="_blank">Fig. 3F</a>, except that 30, 100, 300 and 900 cells per well for bulk and 10, 30 and 100 cells per well for CD45⁺Lin⁻ cells were plated. The results are average of two independent experiments. (E) CD45⁺Lin⁻ ES-HPs were sorted and cultured on OP9 cells with IL-7 and Flt3-L for B cell generation. After one week, cells were harvested and analysed for the expression of B220 and CD19 with flow cytometer. (F) One thousand sorted CD45⁺Lin⁻ ES-HPs were transferred into myeloid differentiation media as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000232#pone-0000232-g001" target="_blank">Fig. 1F</a>. The number of myeloid and erythroid colonies were scored.</p

Characterization of NK progenitors within ES-HPs.

<p>(A) ES-HP cells were stained for the indicated markers and analyzed by flow cytometry. Filled histograms show staining with appropriate mAbs and open histograms show isotype-matched control antibody staining. Percentages of positively stained cells over the control staining are shown. Dead cells were stained with propidium iodide and gated out. OP9 cells were also gated out by their high scatter profile. (B) ES-HPs were co-stained with anti-CD45.2 and IL-7Rα mAbs (left panel), or anti-CD45.2 and γc mAbs (right panel) and analyzed by flow cytometer as in (A). CD45 positive cells were gated and analyzed for the expression of indicated receptors. (C) Irradiated OP9 cells were cultured for 2 days in 96 well plates. ES-HPs were stained with anti-CD45.2 mAb and CD45⁺ and CD45⁻ ES-HPs were sorted by FACS into the wells with the pre-formed OP9 stroma layers. For CD45⁺ ES-HPs, 10, 30 and 100 cells per well were sorted into 12 wells each. For CD45⁻ and bulk ES-HPs, 30, 100 and 300 cells per well were sorted into 12 wells each. After culturing with appropriate cytokines for NK cell differentiation, cells in individual wells were harvested and analyzed for the presence of NK cells by granzyme B RT-PCR. Statistical analysis was performed using L-Calc^TM software. The results are means ± SD of three independent experiments, except for bulk ES-HPs which is average of two experiments.</p

Characterization of ES-HP cells derived from CD34⁺CD45⁻ EBs for surface markers and NK potential.

<p>(A) ES-HP cells derived from CD34⁺CD45⁻ EB cells were stained with mAbs to indicated markers and analyzed by flow cytometry as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000232#pone-0000232-g002" target="_blank">Fig. 2A</a>. (B) ES-HP cells were co-stained with anti-CD45, c-kit and Sca-1 mAbs. Cells were then gated on CD45⁺ and analyzed for the expression of c-kit and Sca-1 (left panel). ES-HP cells were stained with a combination of purified anti-CD45.2 mAb followed by Alexa Flour 647-goat anti-mouse IgG, and biotinylated anti-Mac-1 and Ter119 mAbs followed by streptavidin-PE and analyzed by flow cytometer (middle panel). CD45⁺Mac-1⁻Ter119⁻ cells were gated and analyzed for the expression of IL-2Rβ (right panel). (C) CD45⁺Lin⁻ ES-HPs were sorted and cultured on OP9 stroma with IL-2 and IL-15. After 3 days, cells were harvested and subjected to flow cytometry analysis after staining with anti-IL-2Rβ and anti-IL-7Rα. (D) Sorted CD45⁺Lin⁻ ES-HPs were cultured in NK culture (described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000232#s2" target="_blank">materials and methods</a>) for one week and analysed for the expression of NK markers. (E) The bulk and sorted CD45⁺Lin⁻ ES-HP cells were analyzed for NK progenitor frequency by limiting dilution cultures as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000232#pone-0000232-g002" target="_blank">Fig. 2C</a>. For the former 30, 100 and 300 cells per well and for the latter 3, 10, and 30 cells per well were plated. The results are mean ± SD of three independent experiments.</p

ES-HPs have NK and T cell potentials with little myeloid potential.

<p>(A) ES culture system to generate ES-HPs is illustrated. ES cells are induced to form EBs in methylcellulose. CD34⁺ EBs are sorted and cultured for one week with OP9 stroma in the presence of indicated cytokines to generate ES-HP cells. ES-HPs are cultured on OP9 stroma with IL-2 and IL-15 for NK cell differentiation or on OP9-DL1 stroma with IL-7 and Flt3-L for T cell differentiation. (B) NK cells generated from ES-HPs were stained for the indicated receptors and analyzed by flow cytometer. Filled histograms show staining with the appropriate mAbs and open histograms show isotype-matched control antibody staining. Percentages of positively stained cells over the control staining are shown. Dead cells were stained with propidium iodide and gated out. Residual OP9 cells were also gated out by their high scatter profile. (C) RNA was isolated from 2×10⁵ bulk T cells and 2×10⁶ bulk NK cells derived from ES-HPs (ES-T and ES-NK, respectively) and converted into cDNAs. Aliquots (1/30) of cDNAs were subjected to PCR for rearranged TCRγ gene. The PCR products were blotted and hybridized to a Jγ1 probe. Thymocytes were used as positive control. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) RT-PCR was also used as control. (D) cDNA was generated from ES-T cells as in (C). One microlitre of undiluted, 1/10 and 1/100 diluted cDNA was subjected to PCR for CD3ε and RAG-1. OP9 cells were used as negative control. (E) ES-T cells were harvested on day 7, stained for TCRγδ and TCRβ and analyzed by flow cytometer as in Fig. 1B. OP9-DL1 cells expressed green fluorescent protein and were gated out by green fluorescence. (F) One thousand CD34⁺ EBs and ES-HP cells were plated in methylcellulose media for myeloid and erythroid colony formation and the total number of myeloid and erythroid colonies were counted as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000232#s2" target="_blank">materials and methods</a>.</p

Characterization of CD34⁺CD45⁺ and CD34⁺CD45⁻ EB cells.

<p>(A) EBs were harvested on day 8 and single cell suspension was prepared. Cells were stained with anti-CD34 mAb together with mAb to indicated markers and analyzed by flow cytometry. Dead cells were gated out by propidium iodide staining. (B) CD34⁺CD45⁻ (left) and CD45⁺ (right) EB cells in (A) were gated, and analysed for the expression of Mac-1 by flow cytometry. Numbers indicate percentage of cells in quadrants or gates. (C) CD34⁺CD45⁻ and CD45⁺ EB cells were isolated by FACS sorting and cultured to generate ES-HPs. The sorted EB cells and the ES-HPs generated from them were plated for myeloid and erythroid colony formation as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000232#pone-0000232-g001" target="_blank">Fig. 1F</a>. (D) CD34⁺CD45⁻ EB cells were directly cultured on OP9 stroma with IL-2 and IL-15 for NK cell differentiation. Cells were harvested after 2 weeks, stained for NK markers and analyzed by flow cytometry as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000232#pone-0000232-g001" target="_blank">Fig. 1B</a>. (E) CD34⁺CD45⁻ EB cells were directly cultured onto OP9-DL1 stroma with proper cytokines for T cell differentiation for 3 weeks. Expression of T cell markers was analyzed by flow cytometry as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000232#pone-0000232-g001" target="_blank">Fig. 1E</a>. (F) Limiting numbers (100, 300 and 1,000 cells per well) of CD34⁺CD45⁻ EB cells were sorted into each well of 96 well plates. For NK progenitor assays, wells contained OP9 stroma, IL-2, and IL-15. For T progenitor assays, wells contained OP9-DL1 cells, IL-7 and Flt3-L as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000232#s2" target="_blank">materials and methods</a>. NK cells were detected by granzyme B RT-PCR and T cells detected by TCRγ RT-PCR followed by southern blotting. The number of progenitors in 1000 plated cells was calculated as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000232#pone-0000232-g002" target="_blank">Fig. 2C</a>.</p