Application of small molecule CHIR99021 leads to the loss of hemangioblast progenitor and increased hematopoiesis of human pluripotent stem cells  Yekaterina Galat, Irina Elcheva, Svetlana Dambaeva, Dimantha Katukurundage, Kenneth Beaman, Philip M. Iannaccone, Vasiliy Galat  Experimental Hematology  Volume 65, Pages 38-48.e1 (September 2018) DOI: 10.1016/j.exphem.2018.05.007 Copyright © 2018 Elsevier Ltd Terms and Conditions

Experimental Hematology 2018 65, 38-48. e1DOI: (10. 1016/j. exphem Copyright © 2018 Elsevier Ltd Terms and Conditions

Figure 1 Analysis of the effects of CHIR99021 on the early stages of definitive hematopoietic specification and HE development. (A,B) Schematic representation of two models of definitive progenitor development during hematopoietic hPSC differentiation in vitro. (C) Graph showing the pattern of APLNR and PDGFRα expression in the monolayer CHIR99021 induction differentiation system. (D) Representative flow cytometric analysis demonstrating kinetics of APLNR and PDGFRα expression of CHIR99021-induced and OP9-cocultured hPSCs at day 2 and day 3 of differentiation. (E) Graph comparing CFU-BL and CFU-MS potential of CHIR99021-induced versus OP-cocultured progenitors plated on day 2 and day 3 of differentiation. (F) Representative phase contrast images showing CFUs growing in semisolid medium; MS and BL developed after 3 days of OP9 coculture (left) and MS developed after 3 days in CHIR99021 conditions (right). (G) Graph showing the induction efficiency of CHIR99021-induced versus OP9-cocultured hPSCs as a percentage of CD31+ cells. All experiments were repeated at least three times; the error bars represent SEM. (H) Representative flow cytometric analysis showing the development of CD31+CD34+ and CD31+CD43+ progenitors in OP9 coculture conditions. (I) Representative flow cytometric analysis showing that all of the progenitors that develop in CHIR99021 induction conditions are CD31+CD34+ and lack the expression of CD43+. Experimental Hematology 2018 65, 38-48.e1DOI: (10.1016/j.exphem.2018.05.007) Copyright © 2018 Elsevier Ltd Terms and Conditions

Figure 2 Myeloid and erythroid CFU cell potential assessment of the progenitor cells derived by CHIR99021 induction. (A) Representative flow cytometric analysis showing that the progenitors that arise in CHIR99021 induction conditions are CD31+VE-cadherin (CD144)+ double-positive and develop a substantial CD73– HE fraction. (B) qRT-PCR of RUNX1 expression of progenitor cells at day 5 after CHIR99021 induction. Error bars represent SEM from four independent experiments. (C) Representative flow cytometric analysis of CD43 expression of progenitor cells at day 5 after CHIR99021 induction without (left) or with (right) the addition of VEGF. (D) Representative flow cytometric analysis of CD43 expression of CHIR99021-induced cells after OP9-DLL4 coculture. (E) Representative images of the myeloid hematopoietic colonies developed in semisolid medium after brief OP9-DLL4 coculture. Top: Phase contrast images of colony morphology. Bottom: Corresponding cytospin images (Wright stain). (F) Graph representing relative proportions of the different types of colonies after 16 days in semisolid medium. The graph represents the mean of at least 5 independent experiments. (G) Representative flow cytometric analysis of CD43 and CD235 expression of CHIR99021-derived progenitors after coculture with OP9-DLL4. (H) Graph representing relative proportions of CFU-E and BFU-E that developed in semisolid medium after using the OP coculture (left) or CHIR99021 induction (right) method. The graph represents the mean of at least three independent experiments. Experimental Hematology 2018 65, 38-48.e1DOI: (10.1016/j.exphem.2018.05.007) Copyright © 2018 Elsevier Ltd Terms and Conditions

Figure 3 Floating culture erythroid and NK lymphoid potential assessment of CHIR99021-induced HE progenitor cells. (A) Red blood cell pellet derived from CHIR99021-induced progenitors. (B) Representative flow cytometric analysis of CD45 and CD235 expression of CHIR99021-derived progenitors after coculture with OP9-DLL4 and subsequent culture in erythroid maturation conditions. (C) Wright-stained cytospin images of erythroid cells derived after OP9 induction and CHIR99021 induction. (D) Representative flow cytometric analysis of erythroid culture for markers CD71 and CD36 of cells derived by OP9 induction (top) and CHIR99021 induction (bottom). (E) qRT-PCR of globin expression of erythroid cultures derived using the OP9 coculture induction method. Error bars represent SEM of three independent experiments. (F) qRT-PCR of globin expression of erythroid cultures derived using the CHIR99021 induction method. Error bars represent SEM of three independent experiments. (G) Ratio of HBG and HBB (associated with definitive hematopoiesis) to HBE (associated with primitive hematopoiesis). Error bars represent SEM of three independent experiments. (H) Representative flow cytometric analysis showing CD3 expression gated on the CD15– population. (I) Representative flow cytometric analysis showing the CD34+CD43+CD45– population of CHIR99021-induced cells after coculture with OP9-DLL4. (J) Gating strategy demonstrating the absence of CD14 and CD33 expression within the CD15– cell population of cells analyzed after 3 weeks in lymphoid differentiation conditions. (K) Representative flow cytometric analysis of CD45+ cells gated on the lymphoid fraction of cells analyzed after CHIR99021 induction and compared with OP9 coculture induction. (L) Representative flow cytometric analysis showing the percentage of the CD15– cell fraction. (M) Representative flow cytometric analysis showing the expression of CD56 and CD94 gated on the CD15– population. (N) Gating strategy demonstrating the absence of CD33 expression in the CD15–CD56+ NK cell fraction generated using the CHIR99021 induction method. Experimental Hematology 2018 65, 38-48.e1DOI: (10.1016/j.exphem.2018.05.007) Copyright © 2018 Elsevier Ltd Terms and Conditions

Supplementary Figure S1 IPSC-SR2 Characterization and The Expression Pattern of Early Hematopoietic Specification Markers. (A) IPSC-SR2 pluripotency confirmation by PluriTEST. (B)Normal karyotype of IPSC-SR2. (C) Marker expression during CHIR99021 induced hematopoietic specification. Experimental Hematology 2018 65, 38-48.e1DOI: (10.1016/j.exphem.2018.05.007) Copyright © 2018 Elsevier Ltd Terms and Conditions