Fig. 3: Contribution of heterochronic cell batches to nephron segments and interstitial cells.

a and b Experimental plans showing two strategies for heterochronic recombination of H9 and H9-FP cells, which constitutively express miRFP703 fused to histone H2B. a H9-FP cells were aggregated following directed differentiation and newly differentiated H9 cells were added 2 days thereafter. In b, H9 cells were aggregated following directed differentiation and H9-FP cells were added 2 days thereafter. c and d H2B-marked cell contribution to PODXL+/WT1+ podocytes. e and f H2B-marked contribution to LTL+/HNF4a+ proximal tubules. g and h H2B-marked contribution to CDH1+/BRN1+ distal tubules. i and j H2B-marked contribution to MEIS1+ interstitial cells. Arrowheads in each of the panels indicate H2B-marked cells that have contributed to the labeled cell populations. k–n shows quantification of contribution of H2B marked cells introduced in the batch 1 versus those introduced in batch 2 to each of the labeled cell populations. Cells in three random fields with differentiated structures were counted in each organoid. Values calculated from n = 3 independent biological replicates and expressed as mean ± s.d. Unpaired t test with Welch’s correction was applied to calculate P value. o–q Immunostaining for SIX2, LHX1 on day 18 of differentiation comparing the frequency of SIX2⁺/LHX1⁻ undifferentiated NPCs in single batch versus asynchronous mix organoids. o Example of single batch organoid on day 18. p Example of asynchronous organoid on day 18. q Quantification of cells in single batch versus asynchronous organoids. Cells in 10 random fields were counted in each organoid. Values calculated from n = 3 independent biological replicates and expressed as mean ± s.d. Unpaired t test with Welch’s correction was applied to calculate P value.