1. Journal Club 26/04/2018 Presented By B.HARSHAVARDHAN CeNTAB

2. iPSCs Twenty four candidate genes play pivotal roles in the maintenance of ES cell identity base on their hypothesis. Stable expression of defined factors Ecat1 Dppa5(Esg1) Fbox15 Nanog Eras Dnmt31 Ecat8 Gdf3 Sox15 Dppa4 Dppa2 Fthl17 Sall4 Oct3/4 Sox2 Rex1 Utf1 Tcl1 Dppa3 Klf4 Myc, Kit, Gdf3, Esrrb Oct3/4 NanogSox2 Klf4 iPSCs 2

3. Oct3/4 Involve in the maintenance of self renewal of pluripotent cells. Overexpression leads to the formation of various lineages including primitive endoderm. Oct3/4 Involve in the maintenance of self renewal of pluripotent cells. Overexpression leads to the formation of various lineages including primitive endoderm. Sox2 Essential for embryonic development downregulation by siRNA silencing leads to the differentiation of cell in murine ES cells.Sox2 Klf4 Klf4 repress p53 directly p53 protein suppress Nanog during ES cell differentiation Klf4 contributes to activation of Nanog and other ES cell-specific genes. Klf4 Klf4 repress p53 directly p53 protein suppress Nanog during ES cell differentiation Klf4 contributes to activation of Nanog and other ES cell-specific genes. Nanog Essential for dedifferentiated intermediates to transit to ground state pluripotency. Nanog Essential for dedifferentiated intermediates to transit to ground state pluripotency. iPSCs Potency of Cells Disadvantages Advantages 3

4. Laser Bioprinting 4

5. Matrigel Geltrex Collagen type I from rat tail (3mg/ml) 2 wt% Na-Alg 1 wt% hyaluronic acid Fibrinogen from human plasma (20 mg/mL) Bio-inks 5 Materials

6. 6 Effect of hyaluronic acid on proliferation Printing of hiPSCs Analysis of cell viability, proliferation and metabolic activity after printing Generation and culture of human iPS cells The cell printing system Cell seeding on gel Differentiation ability of cells Pluripotency analysis after printing Methods Gold – 60nm Bio-ink - 50-70um Wavelength – 1064nm Pulse Duration – 10ns Repetition rate – 20Hz Laser Energy – 30-60µJ

7. Microscopic images after 48 hours of hiPSCs and NIH3T3 fibroblasts seeded on layers of different gels Scale bars are 100 μm. Results 7

8. Viability: The viability of hiPSCs 2-3 hours after printing: Printed hiPSCs (82% ± 1%), Non-printed (84% ± 1%), Control cells (87% ± 1%). Results Medium-based approach LDH Assay: Mortality of hiPSCs within two days after printing, By LDH control samples with 100% dead cells Dying cells release lactate dehydrogenase (LDH) into the supernatant medium. MTT assay The metabolic activity of printed, non-printed, and control cells is similar, a bit lower for non-printed cells. Proliferation: The proliferation of printed, non-printed, and control cells showed no systematic differences during a period of four days after printing. 8

9. Bio-ink composed of culture medium only (MED) or hyaluronic acid and culture medium. Printed patterns of hiPSCs on different gels. Scale bars are 200 μm. 9 Results

10. For printing, hiPSCs were suspended in a bio-ink composed of hyaluronic acid and culture medium. The hiPSCs conglomerate as a closer and smaller formation within 24 hours. This aggregation of hiPSCs occurs at different cell quantities per droplet (d, e). Images taken with different microscope magnifications: (a, c) 2.5 x objective; (b, d, e) 5 x objective. Scale bars are 500 μm. Live/dead-staining of printed patterns of hiPSCs on layers of Matrigel™ 4 (a, b) and 24 (c, d, e) hours post-printing. 10 Results

11. Phase-contrast images of the formation of hiPSCs post printing. Printed pattern shortly after printing (left) and formation of tissue-like spheroids (middle) and strands (right) after 6 days. (a2) OCT4 immunostaining and Hoechst 33342 nuclei staining of hiPSCs, printed in discrete droplets 2 days and 6 days and (a3) overlapping droplets 2 days and 6 days post-printing. Formation of tissue-like structures and maintenance of pluripotency after printing with medium and hyaluronic acid on Matrigel. 1000 μm (a2, a3) 500 μm (a1, three middle columns in b and c) 100 μm (left and right detail images in b and c) 11 Results

12. Immunostaining with pluripotency markers (OCT4, NANOG, alkaline phosphatase (ALP), SSEA-4) and proliferation marker Ki67 and cell nuclei staining with Hoechst 33342 as well as merged images (immunostaining + Hoechst 33342) of hiPSCs printed as discrete droplets 1 day. The maintenance of pluripotency is not observably reduced in printed patterns. 12 Results

13. Immunostaining with pluripotency markers (OCT4, NANOG, alkaline phosphatase (ALP), SSEA-4) and proliferation marker Ki67 and cell nuclei staining with Hoechst 33342 as well as merged images (immunostaining + Hoechst 33342) of hiPSCs printed as discrete droplets after 6 days of printing. The maintenance of pluripotency is not observably reduced in printed patterns compared to printed or non-printed cells. 13 Results

14. Spontaneous differentiation of printed and non-printed hiPSCs (medium-based approach, seeded on Geltrex after printing) into derivatives of all the three germ layers ectoderm, endoderm, and mesoderm 21 days after addition of fetal bovine serum. Verification of differentiation via immunostaining with βIII tubulin and cytokeratin 8 for ectodermal, fetoprotein (AFP) and cytokeratin 8 for endodermal, and alpha smooth muscle actin (SMA), von Willebrand (vW), and sarcomeric α- actinin for mesodermal differentiation. Cell nuclei stained with Hoechst 33342. 14 Results Scale bars are 100 μm.

15. (a) Analysis of brachyury expressing mesendoderm progenitor cells. hiPSC were cultured for 2 days in E8 medium after printing. Differentiation into mesendoderm was induced by exposure to 7.5 μM CHIR99021 in RPMI/B27 without insulin for 24 hours. After 48 hours of induction, protein expression of the early mesendoderm marker brachyury was detected by immunofluorescence staining. Directed cardiac differentiation of hiPSCs suspended in a bio-ink composed of hyaluronic acid and culture medium and printed in spot arrays on Matrigel-coated glass slides via temporal small molecule inhibitor modulation of the Wnt/β- catenin signaling pathway. (b) Control for spontaneous differentiation into mesendoderm progenitor cells. Cells were cultured for 6 days in E8 medium after printing. Protein expression of the early mesendoderm marker brachyury (red staining) was analyzed by immunofluorescence staining. Nuclei were stained with 10 μM Hoechst 33342 (blue staining). Scale bars are 1000 μm (overview a, c, d), 500 μm (overview b), 100 μm (e and detailed view in a, b, c, d), and 50 μm (f) 15 Results

16. (e, f) At day 25 of cardiac differentiation (day 29 after printing), protein expressions of the cardiac transcription factor NKX2.5 (red staining) and of cardiac troponin T (cTnT, green staining) were analyzed via immunofluorescence staining. Nuclei were stained with 10 μM Hoechst 33342 (blue staining). Scale bars are 1000 μm (overview a, c, d), 500 μm (overview b), 100 μm (e and detailed view in a, b, c, d), and 50 μm (f) (c, d) At day 10 of cardiac differentiation (day 14 after printing), protein expressions of the cardiac transcription factor NKX2.5 (green staining) and of cardiac troponin T (cTnT, red staining) were analyzed via immunofluorescence staining. 16 Results

17. Directed cardiac differentiation of printed and non-printed hiPSCs via temporal small molecule inhibitor modulation of the Wnt/β-catenin signaling pathway. Cardiomyocyte differentiation was analyzed after 21 days of cardiac induction (25 days after printing) via immunofluorescence staining with cardiac Troponin T (cTnT), cardiac transcription factor NKX2.5, and sarcomeric α-actinin. Cell nuclei were stained with 10 μM Hoechst 33342. Scale bars are 100 μm (overviews), 50 μm (detailed view left), 20 μm (detailed view right). 17 Results

18. Thank You 18