Gene expression of E13 fetal liver of common lymphoid progenitors Flt3+ a4b7+ compared to a4b7-HSAint, a4b7-HSAhi or a4b7-HSAlow. Mus musculus

We used whole genome transcriptome as gene discovery to further understand the rules of lineage restriction in the lymphoid compartment Overall design: Mouse embryos at days 13 gestation were isolated and the fetal livers were dissected. Single cell suspension was prepared and treated with biotinilated antibodies to CD3, CD4, CD8, TER119, Gr1, CD25, CD19, NK1.1 and CD11c. Cell suspensions were then incubated with streptavidin Miltenyi Macs beads. Lineage negative cells were recovered in Macs collumns. Cell suspensions were then incubated with antibodies against Flt3, c-kit, Sca-1, a4b7, HSA and Streptavidin bound to Pacific Blue. Lin-, c-kit+, Flt3+, Sca-1lo, a4b7+ or a4b7-HSAlow,or a4b7- HSAint or a4b7- HSAhi cells were then isolated by cell sorting. 3 biological replicates of each sample were isolated with 5x103 cells. These subsets were shown in single cell diferentiation assays to be have differentpotentials because a4b7+ cells lost B cell potential, while HSA low partially lost B cell potential, HSAhi partially lost T cell potential and HSA int are unbiased in their differentiation potential.

本研究采用全基因组转录组（whole genome transcriptome）作为基因发现策略，以深入解析淋巴区室（lymphoid compartment）内的谱系限制（lineage restriction）规律。实验设计概述：分离妊娠第13天的小鼠胚胎，解剖获取胎肝。制备单细胞悬液，使用针对CD3、CD4、CD8、TER119、Gr1、CD25、CD19、NK1.1及CD11c的生物素化抗体进行孵育。随后将细胞悬液与链霉亲和素标记的Miltenyi Macs磁珠（Miltenyi Macs beads）共同孵育，通过MACS分选柱回收谱系阴性（lineage negative cells，Lin⁻）细胞。再使用针对Flt3、c-kit、Sca-1、α4β7、HSA的抗体，以及标记有太平洋蓝（Pacific Blue）的链霉亲和素对细胞悬液进行孵育。通过细胞分选分离得到Lin⁻c-kit⁺Flt3⁺Sca-1^low的细胞，并进一步分为以下亚群：α4β7⁺细胞、α4β7⁻HSA^low细胞、α4β7⁻HSA^int细胞及α4β7⁻HSA^hi细胞。每份分选的细胞亚群样本设置3次生物学重复（biological replicates），每次获取5×10³个目标细胞。经单细胞分化实验验证，上述细胞亚群呈现出各异的分化潜能（differentiation potential）：α4β7⁺细胞完全丧失B细胞分化潜能，HSA^low细胞部分丧失B细胞分化潜能，HSA^hi细胞部分丧失T细胞分化潜能，而HSA^int细胞则保持无偏向的分化潜能。