Uma Lakshmipathy, Joanna Stella Asprer, Kyle Sylakowski, Chad MacArthur, and Rene Quintanilla Thermo Fisher Scientific, Carlsbad, CA, USA

RESULTS Figure 1. Traditional CytoTune™ reprogramming workflow

(A)Typical workflow CytoTune™ based reprogramming of human fibroblasts, either in feeder based or feeder free cultures, where iPSC colonies are selected on Day 21 post transduction. (B) Reprogramming efficiencies are measured via terminal AP staining of the colonies that have been generated using different reprogramming methods.

Figure 4. Use of combination of positive and negative PSC markers demonstrate full reprogramming

Using the kinetic analysis of SSEA+/CD44- expression patterns in CytoTune™ V2 based reprogramming of human fibroblasts, cells can be enriched via FACS. Cells were sorted into two populations based on their distinct pattern of expression at Day 7. The sorted cells were seeded onto mouse feeders and cultured until day 21 at which time terminal AP staining was performed to demonstrate enrichment of AP+ iPSC colonies.

Figure 8. Utilizing surface marker expression and reprogramming kinetics to enrich for iPSC generation

iPSC generated from human fibroblasts demonstrate very definitive surface marker expression. When stained (feeder dependent) the iPSC stain positively for self renewal markers ( Alkaline Phosphatase Live Stain, SSEA4 and Tra1-60) and demonstrate no CD44 expression (expressed by the surrounding mouse feeders).

ABSTRACT Somatic reprogramming enables the generation of induced pluripotent stem cells (iPSC) from diverse genetic backgrounds. The three week long process is not yet fully understood. To gain a better understanding of the reprogramming process and the differences between different reprogramming systems, it is critical to have the ability to visualize cells as they transition into iPSCs and to track the progression of reprogramming. Current methods for monitoring reprogramming rely on the qualitative inspection of morphology or staining with stem cell-specific dyes and antibodies. Here we present different approaches for using these data for more quantitative analyses. We utilized the IncuCyte™ FLR and ZOOM® Live Cell Imaging System for the continuous monitoring of fibroblasts as they are reprogrammed using different technologies, either in feeder-dependent or feeder-free media systems. The kinetics of colony formation was measured based on confluence in phase contrast images or on fluorescence signals from staining with live Alkaline Phosphatase dye or TRA-1-60 antibody. In addition, whole-well imaging was carried out to derive a more accurate colony count based on traditional AP staining or the more stringent TRA-1-60 antibody staining. The results indicated that the collection of multiple parameters such as colony size and confluence provides semi-quantitative metrics to compare reprogramming differences across the different systems. To further measure the kinetics of reprogramming, flow analysis was performed throughout the process using antibodies against self-renewal markers and fibroblast markers. The combination of real-time visualization and flow analysis enables the quantitative study of reprogramming at different stages and provides a more accurate comparison of different reprogramming systems and methods.

INTRODUCTION The technology of reprogramming somatic cells into Induced Pluripotent Stem Cells (iPSC) has evolved into a easily accessible and widely applicable tool in studying not only basic biology, but also applying its usage towards downstream applications with clinical relevance. We previously identified novel pluripotency tools, like AP Live Stain1 , which can be utilized in the early detection and selection process during reprogramming. During the subsequent usage and analyses of iPSC derived using AP Live stain we performed whole transcriptome analysis of parental somatic cells, established and early stage iPSC, and hESC and found various cell surface markers that showed differential expression in these cell types. One such marker , the cell adhesion protein CD44, was identified and subsequently qualified as a cell surface marker that is highly expressed in human fibroblasts and partially reprogrammed cells, not in hESC and hiPSC2. This negative marker, in conjunction with other early pluripotency markers, such as Alkaline Phosphatase Live Stain, SSEA4 and Tra 1-60 can be used in identifying truly reprogrammed colonies through bio imaging and live cell monitoring during the reprogramming process. In this study we also utilize this differential marker approach , using CD44 and SSEA4, to elucidate the kinetic events during the somatic reprogramming process and how this approach can, with other differentially expressed markers, be potentially used to understand the kinetics of reprogramming.

MATERIALS AND METHODS All materials are Thermo Fisher Scientific reagents, unless stated otherwise Somatic Cell Culture: BJ strain neonatal foreskin fibroblasts (ATCC®) and DF1 adult dermal fibroblasts (donated and collected under all ethical and clinical regulations) were grown in DMEM supplemented with ES-FBS and MEM Non-essential amino acids Reprogramming: CytoTune™ iPSC Sendai Reprogramming kits were used to reprogram fibroblast at an MOI of 3-3-3-3, and CytoTune™ 2.0 iPSC Sendai Reprogramming kits were used at a MOI of 5-5-3, both as per manufacturers suggestions. Reprogramming/ PSC Culture Medias: Feeder dependent iPSC were derived using DMEM/F12 supplemented with KnockOut™ Serum Replacement, MEM Non-essential amino acids, β-mercaptoethanol, and bFGF, grown on mouse feeders. Feeder free iPSC were derived using Essential 8™ media on Geltrex™ LDEV-Free hESC-qualified Reduced Growth Factor Basement Membrane Matrix . Characterization Tools: Reprogramming efficiencies were determined via Vector® Red Alkaline Phosphate terminal staining (Vector Labs). Marker expression was determined using Alkaline Phosphatase Live Stain and antibodies against SSEA4-Alexa Fluor™647, SSEA4-Alexa Fluor™ 488 (BD Biosciences), CD44-Alexa Fluor™488, CD44-PE-Cy®5 and TRA-1-60-Alexa Fluor™488 . Live whole well imaging was performed using the IncuCyte™ ZOOM® (Essen Bioscience). Cell Flow cytometry was performed using the Attune™ Acoustic Focusing Cytometer and analyzed using FlowJo™ V10 (Flow Jo, LLC). FACS was performed on the S3™ Cell Sorter (Bio-Rad Laboratories).

CONCLUSIONS • CD44 and pluripotent surface markers like AP Live Stain, SSEA4

and Tra-1-60 can be utilized to select fully reprogrammed iPSC colonies from partially reprogrammed colonies based differential staining patterns confirming non-overlapping of the pluripotent marker expression and the somatic fibroblast marker expression.

• Reprogramming kinetics tracked through the increased expression of pluripotent markers like SSEA4 and the decreased expression of CD44 can be used to predict the speed and quality of reprogramming.

• Live cell monitoring can be used to look at the morphological changes associated with reprogramming and iPSC colony generation, in addition to surface marker expression.

• Looking at reprogramming kinetics, via cell surface marker expression can help elucidate the timing at which cells can be manipulated for downstream events such as cell enrichment/depletion and for ensuring proper selection of quality cells for cell engineering and other downstream applications

• Further evaluation of early onset pluripotency (SSEA4) and late onset markers (Tra-1-60) against CD44 expression may lead to a better understanding on how different reprogramming systems, media systems, matrices, small molecules and starting somatic population can determine the quality of the reprogramming process

• Live cell monitoring, reprogramming kinetic tracking and enrichment can potentially be applied to other somatic tissues like CD34+ blood cells or PBMCs, which also express CD44 at elevated levels.

REFERENCES (1) Singh U, Quintanilla RH, Grecian S, Gee KR, Rao MS, Lakshmipathy U. Novel Live Alkaline Phosphatase Substrate for Identification of Pluripotent Stem Cells. Stem Cell Rev. 2012 Sep;8(3):1021-9. (2)RH Quintanilla, JST Asprer, C Vaz, V Tanavde, U Lakshmipathy (2014) CD44 is a negative cell surface marker for pluripotent stem cell identification during human fibroblast reprogramming. PLoS ONE 9(1): e85419

ACKNOWLEDGEMENTS We sincerely thank Candida Vaz and Vivek Tanavde, ASTAR, Singapore, for their help with Global Gene Expression analysis.

Real time visualization and kinetic measurement of somatic reprogramming

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Figure 3. Surface markers distinguish reprogramming states

Figure 5. Live Cell Monitoring of reprogramming events

(A) Live monitoring, via IncuCyte™ ZOOM®, of the generation of iPSC colonies from human fibroblasts transduced with CytoTune™ V2 on mouse feeder cells. Representative images of morphological changes (Day7 to Day21) with final surface marker staining of the resulting colonies using the pluripotent markers : alkaline phosphatase live stain, SSEA4, and Tra-1-60 (green) counterstained with the fibroblast marker CD44 (red). (B) Fluorescent imaging of the dual stains demonstrate distinct, non-overlapping expression of positive/ negative markers for fully reprogrammed iPSC colonies Figure 6. Using positive/ negative pluripotency markers expression to track reprogramming kinetics

Figure 7. Dual marker expression can demonstrate differences across reprogramming methods

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Figure 2. CD44 expression is very low in pluripotent cells

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Whole genome analyses of H9 ESCs, BJ Fibroblasts, and iPSC generated from BJs show decreased expression of CD44 in pluripotent stem cells.

Immuno-fluorescent analysis using CD44 and SSEA can distinguish fully reprogrammed iPSC (SSEA4+/CD44-) from partially reprogrammed colonies (SSEA4+/CD44+) during reprogramming (Day21 post transduction).

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© 2015 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientific and its subsidiaries. Essential 8 is a trademark of Cellular Dynamics, International, Inc. CytoTune is a trademark of DNAVECK Corporation. Inccucyte and Zoom are trademarks of Essen Biosciences, Inc. Vector is a trademark of Vector Laboratories. FlowJo is a trademark of FlowJo LLC. S3 is a trademark of Bio-Rad Laboratories, Inc.

(A) The cellular reprogramming is a dynamic process which involves the acquisition of pluripotent marker expression while losing the parental marker expression. (B) Using a positive pluripotent marker (SSEA4) and a negative parental fibroblast marker (CD44) we can observe the dynamic shift in surface marker expression during the feeder free (CytoTune ™ 2.0) reprogramming timeline using flow cytometry.

(A) Tracking SSEA4+ and CD44- expression, throughout the reprogramming process demonstrates differences across reprogramming technologies . Here we observe that CytoTune™ V2 (blue) has a larger population of transduced cells that acquire the SSEA4+ and CD44- marker expression at a much higher rate in comparison to CytoTune™ V1, which correlates to (B) the increased reprogramming efficiency (AP+ colonies) observed at Day 21.

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