Figure 1.
Expression of CD4 on cells co-cultured with or without Notch signaling.
(A) CB and (B) Adult HSCs were maintained in OP9Deltapos (□) or OP9Deltaneg (▪) co-cultures for 28 days with weekly analysis of CD4+ content by flow cytometry. Both CB and adult - derived HSCs expressed CD4 without Notch signaling. Data presented as mean ± SEM (n = 10 for OP9Deltapos and n = 4 for OP9Deltaneg). (C–F) Representative flow cytometry plots showing CD4 and CD14 expression on cells grown in co-culture for 28 days. (C) CB in OP9Deltaneg, (D) CB in OP9Deltapos, (E) adult in OP9Deltaneg, (F) adult in OP9Deltapos.
Figure 2.
Growth of adult and CB cells on OP9Deltapos co-culture.
(A) Representative dot plot (Adult cells, 84 days, 7 days DAPT treatment) showing expression of CD3 and CD4 within the DP population. (B) Plot of CD3 generation by CB (▪) and adult (▵) cells in OP9Deltapos co-culture. (C) Cell number in OP9Deltapos CB (▪) and adult (▵) co-culture (mean ± SEM, n = 9).
Figure 3.
Notch signal interruption promotes CD4 and DP cell differentiation.
CB and adult HSCs were grown in OP9Deltapos co-culture, then Notch signalling was removed for 7 days by transfer to OP9Deltaneg co-culture or addition of DAPT. Cells were analysed by flow cytometry for CD4 and CD8 expression. (A) Representative dot plots for adult cells co-cultured for 70 days. (B–E) Adult (B, D) or CB (C, E) HSCs were grown in OP9Deltapos co-culture (▪) and then either transferred to OP9Deltaneg co-culture (○) or incubated with DAPT (▵) for 7 days. Data points show the percentage of CD4+ ISP (B, C) or CD4+ CD8+ DP (D, E) cells, n≥3, mean+SEM, * p<0.05 for DAPT vs OP9Deltapos (t-test), ** p<0.05 for OP9Deltaneg vs OP9Deltapos (t-test). (F) CD14 (▪) expression plotted against DP expression (▴) for adult cells in OP9Deltapos co-cultures after Notch signalling was removed for 7 days. (G) Cell numbers during DAPT treatment. Paired comparisons of six control and DAPT – treated cultures, showing all HSC-derived cells (left) and DP cells (right).
Figure 4.
Gene expression differences in HSCs co-cultured on OP9Delta cells with or without Notch signalling.
Real-time RT-PCR results from cord blood (CB) or adult HSCs co-cultured with either control OP9Deltaneg (C) cells, or OP9Deltapos (+N) cells. Results are depicted as mean expression ratio +SEM relative to housekeeping gene HPRT (n = 3–4 for CB; n = 2–4 for adults). * p<0.05 compared with ‘d28 +N’ for CB and adult respectively, unpaired 2-tail t-test. d = day. PTCRA = pre-T cell antigen receptor alpha, GATA3 = GATA binding protein 3, IKZF1 = IKAROS family zinc finger 1, TCF7 = Transcription Factor 7 (T-cell specific, HMG-box), HES1 = Hairy and enhancer of split 1, DTX1 = Deltex homolog 1, MAML1 = Mastermind-like 1, CDKN1B = Cyclin-dependent kinase inhibitor 1B.
Figure 5.
Effect of Notch signal removal (DAPT) on mRNA expression of CB and adult HSCs undergoing T lineage differentiation.
Real-time RT-PCR results from cells grown in OP9Deltapos co-culture with Notch inhibitor (DAPT) added for 7 days before harvesting. Timepoints: CB (cord blood) early = 42 days, CB late = 56 days, A (adult) early = 42 days, A late = 70 days. Data are presented as mean fold change in mRNA expression + SEM relative to untreated control cultures (OP9Deltapos), with baseline set at 1.0 (n = 3 for CB; n = 2–4 for adults). * p<0.05 compared with the control, paired 2-tail t-test.
Table 1.
PCR primers.