A Novel Platform to Enable the High-Throughput Derivation and Characterization of Feeder-Free Human ipscs

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1 Supplementary Information A Novel Platform to Enable the High-Throughput Derivation and Characterization of Feeder-Free Human ipscs Bahram Valamehr, Ramzey Abujarour, Megan Robinson, Thuy Le, David Robbins, Daniel Shoemaker and Peter Flynn* Fate Therapeutics Inc., San Diego, CA, USA 1

2 Supplementary Table 1. Composition of SMC4. Small molecule identity, concentration and target of each component in SMC4. Targeted cellular pathway is based on product data sheet. As a supplementation to conventional media, SMC4 is used to generate and culture hipscs. 2

3 Supplementary Table 2. Improved efficiencies in FF reprogramming with SMC4. Reprogramming efficiencies seen with different combination of factors. Table represents ranges across different starting cell lines. *, the emergence of colonies comprising greater than 2 cells. Colony emergence indicates when colonies have emerged and not whether the colonies will develop into bona fide ipscs. **, calculated efficiencies based on the number of SSEA4/Tra181 colonies detected relative to the original number of starting cells. Colonies were scored on day 14 for 4-factor (OKSM) and day 21 for 3-factor (OKS) reprogramming. ***, 1. Sugii, S., Kida, Y., Berggren, W.T. & Evans, R.M. Feeder-dependent and feeder-independent ips cell derivation from human and mouse adipose stem cells. Nat Protoc 6, (2011). 2. Sun, N. et al. Feeder-free derivation of induced pluripotent stem cells from adult human adipose stem cells. Proc Natl Acad Sci U S A 106, (2009). 3

4 Supplementary Table 3. Primer sets. Set of primers used for various studies including qrt- PCR. 4

5 Supplementary Figure 1. Preliminary studies of SMC4. (a) Bright-field images of hipscs seeded on Matrigel coated wells with either conventional medium, ROCKi (conventional medium supplemented with Thiazovivin), MEKi/TGFβi/GSKi (conventional medium 5

6 supplemented with PD , SB and CHIR99021) or ROCKi/MEKi/TGFβi/GSKi (conventional medium supplemented with Thiazovivin, PD , SB and CHIR99021). See Supplementary Table 1 for concentrations. (b) Tra181 expression (red) of hipscs seeded on Matrigel with conventional medium supplemented with various small molecules as (a). Smaller panel located on the bottom right of each image represents DAPI staining (blue). (c) hipscs were seeded at 1 x 10 5 cells on Matrigel with various culture media and scored 4 days later for number of viable cells and percent viability as measured by trypan blue. (d) Comparison of various small molecule inhibitors of ROCK (Thiazovivin at 5μM and Y27632 at 10μM) on seeding and proliferation of hipscs. Colonies cultured with Thiazovivin appear to be more compacted in morphology relative to culture supported by Y (e) Flowcytometry analysis for surface expression of SSEA4 and Tra181 of hipscs cultured in Y27632 or Thiazovivin. (f) IMR90 cells were induced to reprogram using individual virus each expressing one of the 4-factors (Oct4, Sox2, Klf4 and cmyc) and split onto either conventional medium or conventional medium plus ROCKi/MEKi/TGFβi/GSKi (SMC4). Alkaline phosphate staining was conducted on day 20 post infection to identify reprogramming colonies. Brightfield images on the lower right corner are representative of culture in each set. 6

7 Supplementary Figure 2. Viability and differentiation of adapted hipscs. (a) hipscs maintained in either conventional or SMC4 media (FTi60) were assessed for cell death by flow cytometry, as indicated by 7AAD, 4 hours post dissociation into single cells. (b) Three days post removal of SMC4 from the culture medium, FTi60 has changed its morphology to a more differentiated appearance, as indicated by loss of compact colonies and gain of flattened and stretched appearance with increased cytoplasm to nuclei ratio. (c) qrtpcr analysis 14 days post induction of differentiation. Relative gene expression is determined by comparing gene expression of differentiated FTi60 to undifferentiated FTi60 maintained in SMC4. 7

8 Supplementary Figure 3. SMC4 significantly enhances survival during 96-well plate single cell sorting of hipscs in FF culture. SSEA4/Tra181 double positive cells of FTi93 were flowcytometry sorted directly into 96-well plates on FF culture supplemented with SMC4 at various densities. (a) The appearance of early colony formation 24hrs and 48hrs post 96-well sort as indicated by black arrows. (b) Individual colonies on day 8 post sort at various seeded densities in 96-well plates as indicated by black arrows. 8

9 Supplementary Figure 4. Single cell enrichment in combination with SMC4 significantly improves FF reprogramming efficiency of IMR90 fibroblast cells. (a) IMR90 cells on day 8 post infection by individual lentivirus expressing Oct4, Klf4, Sox2 and cmyc were immunoconjugated magnetic beads sorted based on SSEA4 surface marker expression and FF cultured in either conventional medium or SMC4. AP staining was conducted 8 days post culture. The smaller panels in the right-hand corner are representative images of cell/colony morphology. (b) SSEA4/Tra181 double positive cells were scored from three independent experiments. Right panels are representative of a scored colony expressing both SSEA4 and Tra181. (c) Flow-cytometry analysis of derived clone IMR90 SMC4 hipsc after 10 passages in SMC4. (d) Relative qrt-pcr pluripotent gene expression of IMR90 fibroblast cells (IMR90), 9

10 hescs (H1 and HuES9) and hipsc clone IMR90 SMC4 hipsc. Endo, endogenous gene expression. Expression was normalized to Gapdh and relative within each gene group. (e) Relative ectopic gene expression of reprogramming factors in IMR90 fibroblast cells (IMR90), IMR90 fibroblast cells post infected with exogenous genes for 3 days (Day 3 P.I.) and hipsc clone IMR90 SMC4 hipsc. Exo, exogenous expression. Expression was normalized to Gapdh and relative within each gene group. (f) Immunofluorescence staining of clone IMR90 SMC4 hipsc 28 days after EB formation and differentiation. Endoderm, FoxA2; Mesoderm, alpha smooth muscle actin (αsma); Ectoderm, Tuj1. 10

11 Supplementary Figure 5. Generation of FF adipose stem cell derived hipscs. Adipose stem cells were infected with individual lentivirus expressing Oct4, Klf4, Sox2 and cmyc and immunoconjugated magnetic beads sorted based on SSEA4 surface marker expression and FF cultured in SMC4 medium. (a) Bright-field and immunofuorescence staining of adipose stem cell derived hipsc clone (ASC SMC4 hipsc) for pluripotent markers SSEA4 and Tra181. (b) Flow-cytometry analysis of ASC SMC4 hipsc at passage 10. (c) qrt-pcr expression of pluripotent genes of adipose stem cell (ASC), hescs (H1 and HuES9) and ASC SMC4 hipsc. Expression was normalized to Gapdh and relative within each gene group. (d) Immunofluorescence staining of ASC SMC4 hipsc after EB formation and differentiation. Endoderm, FoxA2; Mesoderm, alpha smooth muscle actin (αsma); Ectoderm, Tuj1. 11

12 Supplementary Figure 6. High efficiency in FF reprogramming with 3-factor (polycistronic-oks). (a) Thirteen days after induction, fibroblast cells infected with polycistronic-oks lentivirus were enriched based on surface marker expression by immunoconjugated magnetic beads for SSEA4 and maintained in SMC4. AP staining was conducted on day 30 post induction. (b) Immunofluorescence staining of Tra181 (red) of emerging colonies after enrichment. Left corner panel represent DAPI (blue) staining. 12

13 Supplementary Figure 7. Generation of clonal and FF hipscs using early sort selection. (a) Twelve days post induction with polycistronic-oskm lentivirus, SSEA4/Tra181 positive 13

14 cells were captured and bulk transferred to FF culture with SMC4. Eight days post sort, individual colonies were picked. Images on the right are representative of colonies picked. (b) Picked clones FTi70 and 72 were assessed for SSEA4 and Tra181 expression by flow-cytometry analysis. (c) Cytogenetic analysis of metaphase G-banded cells from clones FTi70 and FTi72 at passage 10. p, passage. (d) EB formation and differentiation of clones FTi70 and 72 after 28 days of differentiation. Endoderm, FoxA2; Mesoderm, alpha smooth muscle actin (αsma); Ectoderm, Tuj1. (e) Fourteen days after polycistronic-oksm lentivirus induction, SSEA4/Tra181 positive cells were flowcytometry sorted and directly transferred to FF 96-well plate at 1 and 3 cells per well. Right panel is a representative image of a colony at day 9 post sort in 96 well-plate; black arrow marks a colony. (f) Sorted clones FTi80 (1 cell per well) and FTi83 (3 cells per well) were assessed for dual expression of SSEA4 and Tra181 by flowcytometry. (g) Cytogenetic analysis of G-banded metaphase cells from clones FTi80 and FTi83 at passage 10. *, 22% of cells displayed nonclonal genomic aberrations but no clonal karyotype detected; **, 44% of cells displayed nonclonal genomic aberrations but no clonal karyotype detected. p, passage. 14

15 Supplementary Figure 8. Characterization of conventionally FTC1 derived clone FTi99. In the initial induction of FTC1 by polycistronic-oks lentivirus used to initiate reprogramming and generation of FTC1 clones 1 and 2, a subset of the infected population was transferred to conventional reprogramming system consisting of conventional medium and feeder cells. Colonies were picked and FTi99 was selected for further analysis. (a) Bright-filed image of a typical culture of FTi99 derived on feeder cells and conventional medium. (b) Immunofluorecence staining of pluripotent markers Oct4, Tra181, Nanog and Tra160 expressed in FTi99. (c) EB formation and differentiation of clone FTi99 after 28 days of differentiation. Endoderm, FoxA2; Mesoderm, alpha smooth muscle actin (αsma); Ectoderm, Tuj1. (d) Cytogenetic analysis of G-banded metaphase cells from clones FTi99 at passage

16 Supplementary Figure 9. Characterization of FTC5 and FTC7 derived clonal hipscs under FF and SMC4 culture. (a) Representative immunofluorecence staining of pluripotent markers Oct4, Tra181, Nanog and Tra160 expressed in individual FTC5 derived hipsc clones 1 16

17 and 2 and FTC7 derived hipsc clones 1 and 2, induced with polycistronic-oks lentivirus and generated using multiplex platform. (b) Representative lineage specific staining of FTC5 and FTC7 derived hipsc, clone 1 and clone 2, respectively, twenty-eight days post the induction of differentiation. Endoderm, FoxA2; Mesoderm, alpha smooth muscle actin (αsma); Ectoderm, Tuj1. (c) Cytogenetic analysis of G-banded metaphase cells from FTC5 and FTC7 derived hipsc clones after long-term FF and single cell culture. p, passage. 17