Injectable biodegradable microcarriers for iPSC expansion and cardiomyocyte differentiation

Abstract Cell therapy is a potential novel treatment for cardiac regeneration and numerous studies have attempted to transplant cells to regenerate the myocardium lost during myocardial infarction. To date, only minimal improvements to cardiac function have been reported. This is likely to be the result of low cell retention and survival following transplantation. This study aimed to improve the delivery and engraftment of viable cells by using an injectable microcarrier that provides an implantable, biodegradable substrate for attachment and growth of cardiomyocytes derived from induced pluripotent stem cells (iPSC). We describe the fabrication and characterisation of Thermally Induced Phase Separation (TIPS) microcarriers and their surface modification to enable iPSC‐derived cardiomyocyte attachment in xeno‐free conditions is described. The selected formulation resulted in iPSC attachment, expansion, and retention of pluripotent phenotype. Differentiation of iPSC into cardiomyocytes on the microcarriers is investigated in comparison with culture on 2D tissue culture plastic surfaces. Microcarrier culture is shown to support culture of a mature cardiomyocyte phenotype, be compatible with injectable delivery, and reduce anoikis. The findings from this study demonstrate that TIPS microcarriers provide a supporting matrix for culturing iPSC and iPSC‐derived cardiomyocytes in vitro and are suitable as an injectable cell‐substrate for cardiac regeneration.


Injectable biodegradable microcarriers for iPSC expansion and cardiomyocyte differentiation
Annalisa Bettini * , Patrizia Camelliti, Daniel J. Stuckey and Richard M. Day * Video 1: Beating iPSC-CM on TIPS microcarriers after 7 days in culture Video 2: Beating iPSC-CM on TIPS microcarriers after 10 days in culture Video 3: Beating iPSC-CM on TIPS microcarriers after 14 days in culture  Table S3: qPCR Primers.Oligonucleotide primers were designed using Primer Blast, to produce a PCR product size of 70-150 base pairs, predesigned to span or flank introns, anneal at 60 o C, <55% GC content and synthesised on a 25 molar scale by Thermo Fisher.

Figure S1 :
Figure S1: Selection of TIPS microcarriers PLGA composition and preconditioning method.(A) Quantification of iPSC attachment to 2%, 5% and 10% 7507 TIPS microcarriers.2.5×10 5 P10 iPSC were seeded on 0.1cm 3 of microcarriers and attached under static dynamic conditions over 24 hours.(B) Comparison of direct-versus post-wetting pre-conditioning of TIPS microcarriers.5×10 5 P7 iPSC were seeded on 0.1cm 3 of microcarriers and attached under static dynamic conditions over 24 hours.Post-wetting coating refers to exposure of the microcarriers to VTN-N, after pre-conditioning.Direct coating refers to exposure of the microcarriers to VTN-N during pre-conditioning.Representative images of iPSC attached to TIPS microcarriers coated (C) directly during the pre-conditioning process, or (D) post-conditioning.Blue arrows show cell attachment to the microcarriers, with formation of cellular connections between microcarriers.Red arrows show cellular clumping and uneven attachment of cells to the microcarriers.Scale bars represent 100 µm.Data are presented as mean ±SD.The significance of the data was calculated by (A) two-way ANOVA, Tukey's post-hoc correction and (B) Unpaired t-test two tailed.(n=6, *P≤0.05,***P≤0.001,****P≤0.0001)

Figure S3 :
Figure S3: Gating strategy utilised to characterise iPSC populations.(A) Selection of live iPSC population by (i) side scatter vs. forward scatter area density plots to remove debris, (ii) side scatter vs. forward scatter height density plots to remove doublets, and (i) selection of live cells by negative expression of dead cell stain.(B) The live cell population was further gated to select the positive population of iPSC expressing (i) SSEA-4, (ii) TRA-1-60, (iii) SOX2 and (iv) OCT4.Representative plots showing gating set according to FMO controls and negative control cells.

Figure S4 :
Figure S4:Gating strategy utilised to characterise iPSC-CMs population.(A) side scatter vs. forward scatter area density plots to remove debris, side scatter vs. forward scatter height density plots to remove doublets, and selection of live cells by negative expression of dead cell stain.(B) Selection of cTNT and ACTN2 positive cell population.Representative plots showing gating set according to unstained and negative control cells.

Figure S5 :
Figure S5: iPSC migration off TIPS microcarriers, and cardiac differentiation.(A) iPSC migrated off TIPS microcarriers onto VTN-N coated tissue culture plastic.Migrated iPSC were differentiated and imaged at (B) day 2 and (C) day 4 of cardiac differentiation.Beating bundles were visible at (D) day 8, and up to (E) day 40 of differentiation.Scale bar represents 100 µm.

Figure S6 :
Figure S6: Impact of colony confluency on iPSC differentiation into iPSC-CM.Images of iPSC 4 days after seeding, at 30%, 50%, 70% and 90% colony confluence.Confluence measurements were performed by visual estimation.The colonies were differentiated into iPSC-CM for 10 days.Optimal confluence for cardiac differentiation was about 30-50%, where cells formed beating bundles from day 6-8 of differentiation.Cardiac differentiation efficiency was quantified by flow cytometric analysis of expression of cardiac markers (A) cTNT and (B) ACTN2.Scale bar represents 100 µm.Data are presented as mean.(n=1-2).

Figure S7 :
Figure S7: Raw western blot film scans used to assess anoikis activation in cellularised TIPS microcarriers samples.iPSC and iPSC-CM attached to TIPS microcarriers were characterised for the by the expression downstream markers of apoptosis, inactive caspases (A) 3 and (B) 9, active cleaved caspase 3. Protein quantification was normalised to the expression of housekeeper GAPDH.Cleaved caspase 3 (cleaved C3).

Figure S8 :
Figure S8: Raw western blot film scans used to assess anoikis activation in iPSC-CM.iPSC-CM controls and suspension samples were characterised for the by the expression downstream markers of apoptosis, inactive caspases (B) 3 and (C) 9, (A) active cleaved caspase 3. (D) Protein quantification was normalised to the expression of housekeeper GAPDH.Negative control (-) refers to cells cultured on 2D tissue culture plastic surfaces, Positive control (+) for the onset of cell death were treated with 100 µM etoposide for 4 hours, Hours (h) refers to the amount of time samples were held in suspension to induce anoikis, Cleaved caspase 3 (cleaved C3).

Figure S9 :
Figure S9: Raw western blot film scans used to assess anoikis activation in iPSC.iPSC controls and suspension samples were characterised for the by the expression downstream markers of apoptosis, inactive caspases (A) 3 and (B) 9, (C) active cleaved caspase 3. (D-E) Protein quantification was normalised to the expression of housekeeper GAPDH.Negative control (-) refers to cells cultured in 2D control conditions, Positive control (+) for the onset of cell death were treated with 100 µM etoposide for 4 hours, Hours (h) refers to the amount of time samples were held in suspension to induce anoikis, Cleaved caspase 3 (cleaved C3).

Figure S10 :
Figure S10: Raw western blot film scans used to assess anoikis activation in remaining iPSC samples.iPSC controls and suspension samples were characterised for the by the expression downstream markers of apoptosis, inactive caspases (A) 3 and (B) 9, (C) active cleaved caspase 3. (D-E) Protein quantification was normalised to the expression of housekeeper GAPDH.Negative control (-) refers to cells cultured on 2D tissue culture plastic surfaces, Positive control (+) for the onset of cell death were treated with 100 µM etoposide for 4 hours, Hours (h) refers to the amount of time samples were held in suspension to induce anoikis, Cleaved caspase 3 (cleaved C3).

Figure S11 :
Figure S11: Flow cytometry assessment of anoikis of iPSC and iPSC-CM preand post-attachment to TIPS microspheres.(A) iPSC and (B) iPSC-CM were cultured in suspension up to 24 hours or attached to TIPS microspheres for 24 hours.iPSC and CM-iPSC attached onto 5 µg/ml VTN-N coated 6-well tissue culture plates were used as negative controls.Positive controls were treated with 10 µM staurosporine for 24 hours.Data are presented as mean ±SD.The significance of the data was calculated two-way ANOVA with Dunnett's post-hoc correction.(n=4-6, *P≤0.05,**P≤0.01,***P≤0.001,****P≤0.0001,statistics to matched negative control).

Figure S13 :
Figure S13: Characterisation of magnetically purified iPSC-CM.iPSC-CM were purified on day 18 of differentiation and marker expression was quantified immediately after purification.(A) Cell count pre-and post-enrichment.(B) Flow cytometric characterisation of the expression of cardiac markers (Ci) cTNT and (Cii) ACTN2 in each purification fraction.The non-CM were collected from the MACS column, as the 1 st fraction.The second step positively selected CMs, flushing out the non-CM.The flushed out non-CM were collected as the 2 nd fraction.Ultimately, the magnetically selected CM, and final product, were collect as the 3 rd and final fraction.Data are presented as mean ±SD.The significance of the data was calculated (A) Unpaired Two-tailed t-test and (B) two-way ANOVA with Sidak's post-hoc correction.(n=4, *P≤0.05,***P≤0.001,****P≤0.0001,statistics to matched negative control).

Figure S14 :
Figure S14: Further assessment of injectable iPSC-CM cellularised TIPS microcarriers.1×10 6 enriched Day 18 iPSC-CM were seeded on 20 mg of microcarriers and incubated under static dynamic conditions for 24 hours.The media was replaced, and the cells were left to recover for another 72 hours.4 days after cell seeding, the sample was resuspended in 600 μL of 60% GranuGel® and injected through a 23G needle capped syringe into a 24 well low-bind plate and the samples were immediately analysed for cell viability or cultured in EB 2% media for an additional 14 days.(A) Viability of iPSC-CM on TIPS microcarriers pre-and postinjection.Percentage cell viability in the sample was quantified using a Chemometec automated cell counter, with viability determined by the use of fluorescent dyes acridine orange and DAPI.(B) Confocal micrograph of iPSC-CM cultured on TIPS microcarriers for 18 days.TIPS microcarrier outlined by dotted grey line.The samples were fixed and stained for nuclear (DAPI blue) and cytoskeleton (phalloidin red) markers.Scale bar represents 50 µm.Data are presented as mean ±SD.n=2.

Figure S15 :
Figure S15: Late iPSC-CM do not expand on TIPS microcarriers.2×10 5 day 18 iPSC-CM were seeded on 20 mg of <250 µm 2% 7507 TIPS microcarriers coated with 5µg/ml VTN-N and attached under static dynamic conditions.Light microscopy images show iPSC-CM attached to TIPS microcarriers after (A) 24 and (B) 48 hours post-seeding.Scale bars represent 100 µm.(C) Quantification of cell attachment suggests that the fraction of cells that attaches the microspheres, does not expand.Data are presented as mean ±SD.The significance of the data was calculated by two-tailed non-parametric t-test with Mann-Whitney post-hoc analysis.(n=3-4, P=0.34).