ABSTRACT. Because pluripotent stem cells (PSCs) can differentiate to any somatic cell type,

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2 ABSTRACT Because pluripotent stem cells (PSCs) can differentiate to any somatic cell type, they are highly studied as a source for cell-based therapies. Two types of PSCs are embryonic stem cells (ESCs) and induced pluripotent stem cells (ipscs). The use of ESCs in clinical applications are limited due to their inherently allogeneic nature and restriction in generating patient-specific or disease-specific cell lines. ipscs hold potential as an autologous cell source for personalized stem cell-based therapies. Mechanical cues have been shown to direct differentiation of PSCs. In vitro studies expand the understanding of how ipscs and ESCs respond to mechanical cues via cytoskeletal proteins. These studies will bring to light the potential role of the cytoskeleton in differentiation. Overall, the objective was to determine the relative cytoskeletal gene expression during differentiation and re-differentiation of ESCs and ipscs, respectively. Spontaneous differentiation and force-mediated differentiation models were used to assess cytoskeletal expression and mesodermal differentiation in PSCs. First, we developed a model of differentiation for ipscs comparable to an established ESC model of differentiation. When spontaneously differentiated as embryoid bodies, ipscs were found to have higher mesodermal and cytoskeletal gene expression than that of ESCs. Under force-mediated differentiation, however, fluid shear stress increased mesodermal gene expression for both ESCs and ipscs but resulted in different cytoskeletal responses. Differences in mesodermal and cytoskeletal expression in ipscs and ESCs prompted further investigation into the cytoskeleton s role during differentiation. More specifically, we analyzed vimentin knockout ESCs (VIM KO-EBs) and wild-type ESCs (ESC-EBs)

3 differentiated in an AggreWell EB model. VIM KO-EBs had lower mesodermal gene expression levels than that of ESC-EBs, indicating that vimentin may play a role in differentiation Overall, these studies indicate that cytoskeletal remodeling of ipscs differed from that of ESCs during differentiation, which may be due to residual properties from the fibroblastic parental cell source of ipscs. In addition, the cytoskeleton may play a role in a cell s differentiation capabilities. Consequently, therapies based on ipscs may need to take into account residual properties from the parental cell source, such as cytoskeletal state and the mechanoresponse.

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6 ACKNOWLEDGMENTS First, I would like to thank my advisor Dr. Tabassum Ahsan for giving me the opportunity to work in her lab, helping me grow and achieve a higher level of thinking, and for her patience over my time in the lab. I would also like to thank Elizabeth Zipf, Kathleen Peucker, and Emma Pineda for their patience when teaching me lab techniques and assisting me in the beginning stages of my project. Thanks also to the previous graduate students in the lab, Russell Wolfe and Kristen Lynch, as well as the current graduate students, Stephanie Messina, Liana Boraas, Michelle Janaszak, Lina Quijano, and Erika Broadnax, and the faculty and staff of the Tulane Department of Biomedical Engineering. ii!

7 TABLE OF CONTENTS ACKNOWLEDGMENTS... ii LIST OF FIGURES... iv LIST OF TABLES... v 1. INTRODUCTION Pluripotent Stem Cells Embryonic Stem Cells Induced Pluripotent Stem Cells Mechanotransduction Cytoskeleton Objective MATERIALS AND METHODS PSC Cell Culture Differentiation Under Static Conditions Application of Fluid Shear Stress AggreWell and Embryoid Body Differentiation Real-time RT-PCR Flow Cytometry RESULTS ipsc Expansion Differentiation Differs with Medium Type Embryoid Body Gene Expression of ESCs and ipscs Pluripotency and Mesodermal Gene Expression Cytoskeletal Gene Expression Mesodermal Gene Expression in Sheared ipscs Cytoskeletal Gene Expression in Sheared ESCs and ipscs Embryoid Body Gene Expression of Vimentin Knockout ESCs DISCUSSION CONCLUSION APPENDIX APPENDIX A: ipsc EXPANSION TRIALS APPENDIX B: ENDODERMAL GENE EXPRESSION OF ESCS AND IPSCS AS EBS APPENDIX C: Gene Expression of Day 12 ESC-EBs and ipsc-ebs APPENDIX D: PLURIPOTENCY AND ENDODERMAL GENE EXPRESSION OF VIMENTIN KNOCKOUT ESC EBS REFERENCES BIOGRAPHY iii!

8 LIST OF FIGURES Figure 1.1. Pluripotent Stem Cell Pluripotency and Differentiation.... 2! Figure 1.2. Sources of pluripotent stem cells... 4! Figure 1.3. Cytoskeletal components of the cell... 9! Figure 2.1. Bioreactor System ! Figure 3.1. ipsc Expansion Protocol ! Figure 3.2. Morphology and Pluripotency of ipscs compared to ESCs... 20! Figure 3.3. Morphology of ESCs and ipscs in two types of differentiation medium ! Figure 3.4. DMEM-based medium had higher mesodermal gene expression than αmembased medium for both ESCs and ipscs ! Figure 3.5. Morphology of ESC-EBs and ipsc-ebs ! Figure 3.6. Pluripotency and mesodermal gene expression of EBs... 27! Figure 3.7. Intermediate filament gene expression of EBs... 29! Figure 3.8. Actin and Tubulin gene expression of EBs ! Figure 3.9. Morphology of ESCs and ipscs after two days of shear stress at 5 dynes/cm ! Figure Mesodermal gene expression of ipscs exposed to shear stress... 33! Figure Cytoskeletal gene expression of ESCs and ipscs exposed to shear stress.. 36! Figure Phase Images of AggreWell VIM KO ESC-EBs ! Figure Phase Images of AggreWell ESC-EBs... 40! Figure Gene expression of ESCs and VIM KO ESCs as EBs ! Figure 4.1. A possible model of de-differentiation and re-differentiation of PSCs... 44! Figure A.1. Morphology of Passage 1 ipscs ! Figure A.2. Pluripotency expression of Passage 1 ipscs... 53! Figure B.1. Endoderm gene expression of ESCs and ipscs as EBs ! Figure C.1. Pluripotency and germ lineage gene expression of ESCs and ipscs as EBs.57! Figure C.2. Cytoskeletal gene expression of ESCs and ipscs as EBs... 58! Figure D.1. Pluripotency gene expression of VIM KO ESCs as EBs ! Figure D.2. Endoderm gene expression of ESCs and VIM KO ESCs as EBs ! iv

9 LIST OF TABLES Table 1. Cytoskeletal Elements and their Functions... 10! Table 2. MEF-iPSC Expansion and number of vials generated !! v

10 1! 1. INTRODUCTION 1.1 Pluripotent Stem Cells Tissue engineering and regenerative medicine approaches offer potential solutions for some of the most pressing medical needs, such as heart disease. In some cell based therapy applications, there is a clinical need for in vitro cell sources. Use of mature adult cell types is limited due to their inability to proliferate for long periods of time and low yields after recovery. Therefore, an alternative cell source is needed: stem cells. Stem cells are generally defined by two properties that distinguish them from all other cells in the body: their capacity for long-term self-renewal and their ability to differentiate into specialized cells in vivo and in vitro. Pluripotent stem cells (PSCs) have become important and useful in understanding the mechanisms of diseases and treating patients with various pathologies due to their potential to differentiate into any type of specialized cell (Figure 1.1) [1]. Thus, PSCs, which include embryonic stem cells (ESCs) and induced pluripotent stem cells (ipscs), can become all cells in the body.

11 2! Figure 1.1. Pluripotent Stem Cell Pluripotency and Differentiation. Pluripotent stem cells have the ability to self-renew and differentiate into the three germ layers (endoderm, mesoderm, and ectoderm), from which arise all the cells in the body.

12 3! 1.2 Embryonic Stem Cells Embryonic stem cells (ESCs) provide useful research tools due to their pluripotent potential and regenerative abilities. ESCs are derived from the inner cell mass (ICM) of the pre-implantation blastocyst (Figure 1.2A). The ICM gives rise to the entire body of the organism through the development of the three germ lineages (endoderm, mesoderm, ectoderm) [2]. In vitro, ESCs are plated and expanded on mitotically inactivated feeder layers, such as mouse embryonic fibroblasts (MEFs), to facilitate selfrenewal and maintenance of pluripotency. When cultured without a feeder layer but in the presence of leukemia inhibitory factor (LIF), mouse ESCs also remain pluripotent and self-renew [2, 3]. When LIF is removed, however, mouse ESCs undergo spontaneous differentiation [4]. Thus, ESCs are a potential cell source for all somatic cell types. Even though ESCs have provided numerous insights into certain diseases and cell function, the use of human ESCs in clinical applications are limited due to their inherently allogeneic nature and restriction in generating patient-specific or disease-specific cell lines.

13 4!! Figure 1.2. Sources of pluripotent stem cells. (A) ESCs are derived from the inner cell mass of the pre-implantation blastocyst and then plated and expanded in vitro. (B) Adult somatic cells, such as fibroblasts, are obtained from the donor and reprogrammed by the exogenous addition of sets of transcription factors (e.g. Oct3/4, Sox2, Klf4, and c-myc) to generate ipscs.

14 5! 1.3 Induced Pluripotent Stem Cells Induced pluripotent stem cells (ipscs) hold potential as an autologous cell source for personalized stem cell-based therapies. By reprogramming the patient s own somatic cells into a stem cell-like state similar to ESCs, autologous cell-based therapies with ipscs benefit from a lower chance of immune rejection. Takahashi and Yamanaka developed this technique by reprogramming through the retroviral delivery of four transcription factors: Oct3/4, Sox2, Klf4, and c-myc [5]. Using such a transfection, ipscs can be generated from multiple cell types including mouse embryonic fibroblasts (MEFs) (Figure 1.2B). ipscs resemble ESCs in certain ways including their morphology, proliferation, gene expression, and potential to form teratomas [1, 6]. Studies have found differences compared to ESCs, however, in ipsc potential to differentiate to all cell types [7], their epigenetic memory [8], and their gene expression profiles [9]. These differences could be due to an inefficient reprogramming process or residual properties from the parental cell source of the ipscs [10]. Although ipscs show many similarities to ESCs, a more thorough understanding of the differences are needed to develop effective clinical applications using pluripotent stem cells.

15 6! 1.4 Mechanotransduction Mechanotransduction is the process of converting mechanical signals into biochemical responses, which influence cell behavior and function [11]. Numerous signaling and structural molecules, including integrins, cell-cell adhesion molecules, growth factor receptors, mechanosensitive ion channels, and cytoskeletal filaments, contribute to mechanotransduction responses [12]. Studying cellular contractility, stiffness of surrounding tissues, and resulting mechanical deformations and stresses offers insight into how mechanical forces regulate cell proliferation and differentiation in vitro and in vivo [13]. In addition, the physical microenvironment provides signals that regulate many developmental processes. For example, studies have shown that cellular deformation due to mechanical forces may be a regulator of early stem cell differentiation [11]. Mechanical linkages, such as through the cytoskeleton, allow force transfer across the cell regulating biochemical signals in the cytoplasm and transmitting force to the nucleus. Therefore, characterizing and understanding the factors that regulate the cytoskeleton is critical in understanding cell fate. 1.5 Cytoskeleton The cytoskeleton serves as the main dynamic, structural framework of a cell carrying out many functions including organization of the cell, physical and biochemical connection to the external environment, and force generation which enables cell movement and change of shape. Microtubules, microfilaments, and intermediate filaments comprise the three major polymer components of the cytoskeleton. All three

16 7! components interconnect with each other to control the shape and mechanical integrity of a cell (Figure 1.3). These filament systems differ structurally and functionally in many ways, such as in mechanical stiffness, assembly dynamics, polarity, and associated molecular motors [14], which all contribute to the properties of the cytoskeleton as a whole. The three types of filaments possess some similar and distinct properties (Table 1). Microtubules, comprised of tubulins, provide the stiffest filaments, playing a role in cell division [15], transport of intracellular components [14], and maintenance of cell shape [16]. Microfilaments, or actin filaments, are less rigid than microtubules, but are highly organized due to the high concentration of crosslinkers binding to actin filaments. The linkages promote the assembly of isotropic networks, bundled networks, and branched networks [14]. Actin microfilaments have many functions including providing mechanical support to cells, linkages to other components, and cell motility. Actin filaments continually assemble and disassemble producing the physical force needed for filopodial or lamellipodial protrusions and readjustment of adhesive contacts, or focal adhesions, to the cell environment. These physical forces are required for motile cell functions, such as chemotaxis, cell-cell communication, and cell-matrix binding [17]. Specifically, smooth muscle cell actin aids in contractility of the cell. Through these microfilaments, actomyosin contractility contributes to nuclear movement in cells, such as in leukocytes and neuronal cells [18]. Both microtubules and microfilaments are polarized polymers, resulting in suitable tracks for molecular motors, which have crucial roles in organizing these polymers.

17 8! Conversely, intermediate filaments are non-polar and the least stiff of the cytoskeletal polymers. Intermediate filaments absorb mechanical stresses that contribute to cell stiffness and cell shape [19]. The intermediate filament lamin lines the nuclear envelope. More specifically, A-type lamins were assessed in this study due to their role in mechanical stability and regulation of cellular processes, such as nuclear positioning, gene transcription, DNA replication, DNA damage response, cell differentiation, and cell polarization during migration [20]. Nestin, another important intermediate filament, contributes to cytoskeletal remodeling by regulating the assembly and disassembly of intermediate filaments and other proteins [21]. Nestin also serves as a marker for central nervous system progenitor cells. Keratin intermediate filaments maintain the structural integrity of epithelial cells and act as an intracellular scaffold contributing to cell stiffness and withstanding external mechanical forces [22]. Lastly, the intermediate filament vimentin increases the mechanical integrity of cells while localizing and stabilizing intracellular organelles [23, 24]. In addition, vimentin plays an essential role in cell shape, focal adhesions, and motility changes occurring during epithelial-mesenchymal transition [25]. Together, all three cytoskeletal components contribute to the entire cytoskeleton network. While the function of the cytoskeletal proteins has been studied in a wide range of context, their role in stem cell differentiation still needs to be expanded.

18 9! Figure 1.3. Cytoskeletal components of the cell. The cytoskeletal elements (microtubules, intermediate filaments, and actin filaments) span the cytoplasm and serve as a link between the extracellular space and the nucleus.

19 10 Table 1. Cytoskeletal Elements and their Functions.

20 Objective Thorough analysis of the role of the cytoskeleton of ipscs compared to the standard ESCs would allow further understanding of their mechanoresponse in vivo. These studies will bring to light the potential role of the cytoskeleton in differentiation. Overall, the objective was to determine the relative cytoskeletal gene expression during differentiation and redifferentiation of ESCs and ipscs, respectively. Due to incomplete reprogramming of somatic cells to a stem cell state, ipscs may have residual properties from their parental cell source. Our overarching hypothesis was that residual properties from the parental fibroblast cell source of ipscs would alter cytoskeletal expression and mesodermal differentiation of ipscs compared to ESCs. Further, we hypothesized that the differences in mesodermal differentiation were the result of cytoskeletal differences indicating that the cytoskeleton plays a role in differentiation. To evaluate this hypothesis, we proposed the following aims. Aim 1. Determine changes in cytoskeletal expression in PSCs during spontaneous differentiation. Aim 2. Determine changes in cytoskeletal expression in PSCs after force-mediated differentiation. Aim 3. Analyze the role of vimentin during spontaneous differentiation.

21 12 2. MATERIALS AND METHODS 2.1 PSC Cell Culture Mouse ES-D3 embryonic stem cells were obtained from ATCC (Manassas, VA), and mouse embryonic fibroblast (MEF)-derived induced pluripotent stem cells (ipscs) were obtained from Stemgent (Cambridge, MA). The ESCs or ipscs were expanded on mitotically-inactivated MEFs or gelatin-coated T175 flasks. The culture medium for both ESCs and ipscs consisted of Dulbecco s Modification of Eagle s Medium (DMEM) supplemented with 15% ES-qualified fetal bovine serum (Invitrogen), 2mM L-glutamine, 100U/ml penicillin/streptomycin, 0.1mM non-essential amino acids, 0.1mM betamercaptoethanol, and 1000U/ml leukemia inhibitory factor (LIF: Fisher Scientific). This medium was changed daily during expansion. 2.2 Differentiation Under Static Conditions ESCs and ipscs were differentiated on acid etched, collagen type IV-coated glass slides in culture medium but without LIF. Collagen type IV (BD Biosciences, Bedford, MA) at a concentration of 3.5 µg/cm 2, diluted with 0.05M HCl, was used to coat the slides for one hour. ESCs seeded at 10,000 cells/cm 2 or ipscs seeded at 20,000 cells/cm 2 and 40,000 cells/cm 2 were incubated at 37 C/5% CO 2 in either DMEM-based medium or αmem-based medium (25ml) for four days. DMEM-based medium consisted of DMEM supplemented with 15% ES-qualified fetal bovine serum (Invitrogen), 2mM L-glutamine, 100U/ml penicillin/streptomycin, 0.1mM non-essential amino acids, and 0.1mM betamercaptoethanol. αmem-based medium consisted of Alpha Minimum Essential Medium

22 13 (αmem), 10% fetal bovine serum, 100U/ml penicillin/streptomycin, and 0.1mM betamercaptoethanol. The medium was changed at day two of differentiation. 2.3 Application of Fluid Shear Stress ESCs and ipscs were differentiated on acid etched, collagen type IV-coated glass slides in αmem-based medium. Collagen type IV (BD Biosciences, Bedford, MA) at a concentration of 3.5 µg/cm 2, diluted with 0.05M HCl, was used to coat the slides for one hour. ESCs seeded at 10,000 cells/cm 2 or ipscs seeded at 40,000 cells/cm 2 were incubated at 37 C/5% CO 2 in αmem-based medium (25ml) for a pre-treatment duration of two days. Fluid shear stress was applied to the ESCs or ipscs using a parallel plate bioreactor system. The closed-loop bioreactor system consisted of a plastic bottle connected with tubing to a dampener and a flow chamber. The flow chamber contained a flow block above a spacer that sits on a glass slide, allowing a channel for fluid flow, all held together by an aluminum frame on top and bottom with a rubber gasket to prevent leaks (Figure 2.1). A Masterflex peristaltic pump was used to recirculate medium through the loop system and allow for steady laminar shear stresses of 1.5, 5.0, or 15 dynes/cm 2 for two days.

23 14 Figure 2.1. Bioreactor System. The bioreactor system (left) uses a peristaltic pump to recirculate medium from a bottle, through a pulse dampener, across a flow chamber, and back again (schematic in middle). The flow chamber is an assembly of a flow block above a spacer that sits on a glass slide, all held together by a top and bottom aluminum frame with a gasket to prevent leaks (schematic on left). [Wolfe et al., ES Cells: Methods and Protocols]

24 AggreWell and Embryoid Body Differentiation ESCs and ipscs were allowed to differentiate as embryoid bodies for up to 12 days. After pluripotent cells were expanded, cells were placed in 100mm non-tc treated petri dishes (0.5x10 6 cells/10 ml) in culture medium without LIF. The petri dishes were placed on a rotary shaker (40RPM, New Brunswick ) in an incubator and kept in constant motion. The medium and dishes were changed daily using gravity separation after the second day. ESCs and vimentin knockout (VIM KO) ESCs were differentiated in AggreWells (800 cells/eb) in culture medium without LIF with daily medium changes. After 24 hours, ESC-EBs were transferred from the AggreWells to non-tc treated petri dishes (1 AggreWell/petri dish) in culture medium without LIF. After 4 days, VIM KO-EBs were transferred from the AggreWells to agar-coated petri dishes (1AggreWell/petri dish) in culture medium without LIF. As described above, the petri dishes with ESC-EBs were kept in constant motion on a rotary shaker, while the VIM KO-EBs on agar-coated petri dishes were statically cultured. The medium and dishes were changed daily using gravity separation. 2.5 Real-time RT-PCR All cell samples used for PCR were prepared using the same protocol. Samples were trypsinized to create cell solutions, centrifuged, aspirated, and lysed using lysis buffer made with RLT buffer (Qiagen RNeasy Mini Kit) and beta-mercaptoethanol. RNA was isolated and quantified using a Nanodrop spectrophotometer for each cell sample. cdna converted from 1µg RNA (Invitrogen Superscript III First-strand synthesis) was

25 16 used to analyze mrna levels using SYBR Green (Applied Biosystems, Carlsbad, CA) on a StepOnePlus TM PCR system. Gene expression levels, quantified using the standard curve method, were normalized to the housekeeping gene GAPDH. 2.5 Flow Cytometry Cell solutions were generated using StemPro Accutase (Invitrogen, Carlsbad, CA) and subsequently fixed with 4% formaldehyde for 15 minutes at 4 C. The cells were then stored in working buffer solution, which consisted of PBS (with calcium, with magnesium), 0.3% bovine serum albumin, and 0.001% polyoxyethylenesorbitan monolaurate, at 4 C. Samples analyzed for intracellular markers were permeabilized using 0.5% triton-x (Sigma-Aldrich). All samples were blocked with 10% serum solution and then stained with a primary antibody. A secondary antibody stain was used if necessary to detect fluorescence. Relative protein expression was analyzed using an Attune Acoustic Focusing Cytometer and data was analyzed using FCS Express software.

26 17 3. RESULTS 3.1 ipsc Expansion Expansion of ipscs was an important process to generate a cell bank for lab use (Table 2). Passage 0 mouse embryonic fibroblast (MEF)-derived ipscs were obtained from Stemgent (Cambridge, MA). The developed protocol for the ipsc expansion generated a substantial population of ipscs that could be used for experimental testing (Figure 3.1). First, MEFs were cultured and Mitomycin-C treated to generate a mitotically inactive feeder layer for the expansion of ipscs. Passage 0 ipscs were cultured for two days and split into 36 vials upon freezing (now Passage 1). After initial studies, the developed protocol involved seeding one-eighth of a vial of Passage 1 ipscs onto a gelatin-coated flask cultured for 6 days in culture medium. These cells were then trypsinized and split 1:5 (now Passage 2) onto gelatin-coated flasks. Passage 2 cells were cultured for 5 days and frozen down at approximately 300,000 cells/vial (Passage 3). Passage 3 ipscs generated were similar in morphology to pluripotent ESCs, both having rounded, dome-like colonies and refractive edges (Figure 3.2A). Using flow cytometry, ipsc protein expression of the pluripotency marker NANOG was similar to that of ESCs. Both ESCs and ipscs had fluorescence that was higher (shifted to the right) compared to their secondary controls. Further, the expression was at similar levels for both ESCs and ipscs (Figure 3.2B). Thus, based on these results, we expect that the banks of expanded ipscs remained pluripotent.

27 18! Table 2. MEF-iPSC Expansion and number of vials generated.!

28 19 Figure 3.1. ipsc Expansion Protocol. Mitomycin-C Treatment of MEFs was used to obtain a mitotically inactive feeder layer for Passage 0 ipscs to be seeded on. Passage 0 cells were frozen down into 36 vials. 1/8 vial of Passage 1 ipscs were seeded on gelatin, cultured for 6 days and split 1 to 5 flasks. Passage 2 cells were cultured for 5 days then frozen down into 72 vials.!

29 20 Figure 3.2. Morphology and Pluripotency of ipscs compared to ESCs. (A) Phase images of ESCs and ipscs after culture for 4-5 days on gelatin. (B) NANOG protein expression represented in histograms, where ESCs are in black and ipscs are in red, along with secondary only staining controls. Scale bar represents 200µm.

30 21! 3.2 Differentiation Differs with Medium Type An adherent model of differentiation needed to be developed in ipscs to compare differentiation capabilities of ipscs to ESCs. ipscs were seeded at different densities onto collagen type IV-coated glass slides and differentiated for four days under static conditions to determine an appropriate seeding density for subsequent studies. Previously, a seeding density of 10,000 cells/cm 2 for ESCs has been suitable for 2D differentiation and shear stress studies [11]. ipscs seeded at 20,000 and 40,000 cells/cm 2 were analyzed. A seeding density of 40,000 cells/cm 2 was selected for future experiments due to similarities to ESC samples in the morphology, confluence, and refractivity (Figure 3.3). These seeding densities were tested in DMEM-based medium and αmembased medium to examine any differences in the two types of differentiation mediums. After four days of differentiation on glass slides, the mesodermal markers T-BRACHY and FLK1 had higher expression levels in DMEM-based medium compared to αmembased medium in ESC samples. A similar trend was observed in ipsc samples seeded at 20,000 and 40,000 cells/cm 2 (Figure 3.4). αmem-based medium was used in subsequent experiments to maximize detection of small increases in differentiation due to applied shear stress.

31 22! Figure 3.3. Morphology of ESCs and ipscs in two types of differentiation medium. ESCs and ipscs were seeded at different densities on collagen type IV-coated glass slides and allowed to differentiate using DMEM-based medium or αmem-based medium for four days in static conditions. Scale bar represents 200µm.

32 23!! Figure 3.4. DMEM-based medium had higher mesodermal gene expression than αmem-based medium for both ESCs and ipscs. Gene expression of T-BRACHY (TOP) and FLK1 (BOTTOM) for ESCs and ipscs in DMEM-based medium compared to αmem-based medium.

33 Embryoid Body Gene Expression of ESCs and ipscs Pluripotency and Mesodermal Gene Expression Using an embryoid body (EB) culture model allowed for a method of analyzing the differences between PSCs in their ability to spontaneously differentiate. ESCs and ipscs were spontaneously differentiated as EBs for up to 8 days and assessed for morphology, pluripotency, and mesodermal gene expression to validate that these PSCs differentiate and lose pluripotency. The parental source of ipscs, originating from a fibroblastic (mesoderm) cell source, motivated the analysis of mesodermal (re)differentiation. Both ESC-EBs and ipsc-ebs formed rounded cell clusters. Furthermore, with increased culture duration, the edges of all EBs became sharper (Figure 3.5). This is commonly seen in EB cultures and is an indicator of the formation of a surface epithelial layer. In addition, it is clear that cells within the EBs are proliferating, as there is an observed increase in EB size between day 4 and day 8. With increased culture duration, pluripotency (NANOG) gene expression decreased with a similar declining trend for ESC-EBs and ipsc-ebs with no significant difference at day 8 based on cell type (Figure 3.6A). For the mesodermal marker T-BRACHY, ESC-EBs and ipsc-ebs had transient expression decreasing to low levels at day 8, with ipsc-ebs at a significantly (p<0.05) lower level than ESC-EBs (Figure 3.6B). Expression of FLK1, another mesodermal marker, steadily increased over time, with ipsc-ebs at a significantly (p<0.01) higher expression level than ESC-EBs at day 8 (Figure 3.6C). Increased mesodermal expression in ipsc-ebs compared to ESC-EBs demonstrates the

34 25 possibility of ipscs differentiating toward the lineage of their fibroblastic cell source at a faster rate than ESCs. Thus, spontaneous differentiation resulted in a loss of pluripotency for both ESCs and ipscs, and ipsc-ebs seemed to have higher mesodermal gene expression compared to ESC-EBs over time.!

35 26!! Figure 3.5. Morphology of ESC-EBs and ipsc-ebs. Phase Images of ESC (LEFT) and ipsc (RIGHT) after four days (TOP) and eight days (BOTTOM) of differentiation in suspension culture as EBs. All images are taken at the same magnification with the scale bar indicating 200µm.

36 27! Figure 3.6. Pluripotency and mesodermal gene expression of EBs. ESCs and ipscs were differentiated as EBs and assessed for gene expression at day 0, 2, 4, and 8 for pluripotency (NANOG) and mesoderm markers (T-BRACHY and FLK1). Data presented are averages (mean ± SEM, n = total of 3 replicates from 1 trial), while day 0 analysis is of a single sample. Significant differences between ESC-EB and ipsc- EB samples at day 8 are indicated by asterisks (* p<0.05 and ** p<0.01).

37 28! Cytoskeletal Gene Expression Reprogramming of ipscs to a pluripotent cell state causes the cells to lose their extensive cytoskeleton. However, ipscs may still contain residual properties from their parental source, causing a higher initial expression than ESCs at the stem cell state. Redifferentiation then results in the reformation of the cytoskeleton for ipscs. Therefore, assessing cytoskeletal gene expression of ipscs compared to ESCs after differentiation would allow for a better understanding of ipscs re-differentiation capabilities. ESCs and ipscs were differentiated as embryoid bodies (EBs) for up to 8 days. For both ESC-EBs and ipsc-ebs, VIM (Figure 3.7A), LMNA (Figure 3.7B), and ACTA2 (Figure 3.8A) gene expression steadily increased over time with ipsc-eb levels significantly (p<0.01, p<0.05, and p<0.01, respectively) higher than that of ESC-EBs at day 8. KRT8 expression also increased for both ESC-EBs and ipsc-ebs over time but with no detectable difference at day 8 based on cell type (Figure 3.7C). ESC-EBs and ipsc-ebs both expressed fairly level NES (Figure 3.7D) and TUBA1B (Figure 3.8B) expression with differentiation, but ipsc-eb levels were significantly (p<0.01) higher than that of ESC-EBs at day 8. After 8 days of spontaneous differentiation, ipsc-ebs had higher cytoskeletal gene expression levels compared to ESC-EBs, similar to the trend seen in mesodermal markers. Thus, higher levels of cytoskeletal expression and mesodermal differentiation in ipsc-ebs may result from residual properties from their parental cell source.!

38 29! Figure 3.7. Intermediate filament gene expression of EBs. ESCs and ipscs were differentiated as EBs and assessed for gene expression at day 0, 2, 4, and 8 for the intermediate filaments VIM, LMNA, KRT8, and NES. Data presented are averages (mean ± SEM, n = total of 3 replicates from 1 trial), while day 0 analysis is of a single sample. Significant differences between ESC-EB and ipsc-eb samples at day 8 are indicated by asterisks (* p<0.05 and ** p<0.01).!

39 30!! Figure 3.8. Actin and Tubulin gene expression of EBs. ESCs and ipscs were differentiated as EBs and assessed for gene expression at day 0, 2, 4, and 8 for smooth muscle cell actin (ACTA2) and tubulin (TUBA1B). Data presented are averages (mean ± SEM, n = total of 3 replicates from 1 trial), while day 0 analysis is of a single sample. Significant differences between ESC-EB and ipsc-eb samples at day 8 are indicated by asterisks (** p<0.01).

40 Mesodermal Gene Expression in Sheared ipscs After assessing spontaneous differentiation of both ESCs and ipscs, a controlled, or directed, differentiation model was used to further understand the cytoskeletal response of these cell types to force-mediated differentiation. The cytoskeleton is an important contributor to mechanotransduction by allowing force transfer across the cell regulating biochemical signals and transmitting force to the nucleus. Differences in cytoskeletal and mesodermal gene expression found among cell types led to the question of how each cell type responds to mechanical stimulation. When shear stress is applied to a cell, the cytoskeleton transmits forces to the nucleus, further modulating gene expression and ultimately promoting differentiation. Effects of shear stress were assessed on mouse ESCs and ipscs that were allowed to differentiate on collagen type IV-coated glass slides for an initial period of two days followed by two days of fluid shear stress of 1.5, 5.0, or 15 dynes/cm 2. Within each experiment, samples cultured with a matched volume of medium but under static conditions served as controls. Compared to one another, ESCs and ipscs had similar morphology, confluence, and refractivity for static controls, as well as samples exposed to a stress of 5.0 dynes/cm 2 (Figure 3.9). Further, expression of FLK1, a mesodermal marker, significantly (p<0.01) upregulated in ipscs when exposed to 5.0 dynes/cm 2 (Figure 3.10). This is similar to our finding with ESCs, where expression of various mesodermal markers, including FLK1, increased with applied shear stress. Thus, force-mediated differentiation resulted in an upregulation in mesodermal expression for both ESCs and ipscs, validating that applied shear stress promotes differentiation in both phenotypes.

41 32 Figure 3.9. Morphology of ESCs and ipscs after two days of shear stress at 5 dynes/cm 2. Phase Images of ESCs (TOP) and ipscs (BOTTOM) after two days of shear stress (RIGHT) compared to static controls (LEFT). Scale bar represents 200µm.!

42 33 Figure Mesodermal gene expression of ipscs exposed to shear stress. FLK1 gene expression in samples cultured as static controls (white bars) or exposed to 2 days of 5.0 dynes/cm 2 of shear stress (black bars). Data represented are averages (mean ± SEM,, n = 4). The asterisks indicate a significant difference between static and shear samples (** p<0.01).

43 Cytoskeletal Gene Expression in Sheared ESCs and ipscs Using the same directed differentiation model, the cytoskeletal elements were analyzed to assess the cytoskeleton s response to applied fluid shear stress in both phenotypes. Cytoskeletal genes evaluated were the intermediate filaments vimentin (VIM), lamin A (LMNA), keratin 8 (KRT8), and nestin (NES), as well as smooth muscle cell actin (ACTA2). Regarding ESCs, VIM and LMNA gene expression were significantly (p<0.01 or p<0.05) upregulated when exposed to shear stresses of 1.5 or 5.0 dynes/cm 2 (Figure 3.11A, B, respectively). Yet, a higher stress magnitude of 15 dynes/cm 2 had no detectable effect for both genes. KRT8 and NES expression, however, was significantly (p<0.05 or p<0.01) upregulated for all magnitudes of applied shear stress (Figure 3.11C, D, respectively). Conversely, smooth muscle cell actin (ACTA2) gene expression did not detectably change in any of the tested conditions (Figure 3.11E). Thus, this study on ESCs found that gene expression of intermediate filaments, but not actin microfilaments, changed in response to applied stress. To determine the effect of applied shear stress on ipscs, these cells were exposed to a shear stress of 5.0 dynes/cm 2, which had induced a significant response for all intermediate filaments tested in ESCs. For ipscs, applied shear stress did not detectably affect gene expression of the intermediate filaments VIM, LMNA, KRT8, and NES (Figure 3.11A, B, C, D, respectively). This magnitude of shear stress, however, did induce a downregulation in gene expression of ACTA2 (Figure 3.11E). For all genes tested, ipsc static groups had considerably higher gene expression compared to ESC shear groups. These higher expression levels in ipscs may be due to residual properties from the previously developed cytoskeleton of the fibroblastic cell source. Thus,

44 35 application of shear stress at levels that were found to induce changes in intermediate filaments for ESCs had no effect in ipscs. While this directed differentiation model caused similar increased mesodermal gene expression for both ESCs and ipscs, different cytoskeletal responses were found between the two phenotypes.

45 36!! Figure Cytoskeletal gene expression of ESCs and ipscs exposed to shear stress. Gene expression in samples cultured as static controls (white bars) or exposed to 2 days of 1.5, 5.0, or 15 dynes/cm 2 (black bars). Data represented are averages (mean ± SEM,, n = total of 4-6 replicates from 1 or 2 independent trials). Significant differences between static and shear samples are indicated by asterisks (* p<0.05 and ** p<0.01).

46 37! 3.5 Embryoid Body Gene Expression of Vimentin Knockout ESCs Spontaneous differentiation and force-mediated differentiation caused different mesodermal and cytoskeletal responses in ipscs and ESCs, prompting further investigation into the cytoskeleton s role during differentiation. More specifically, the role of the intermediate filament vimentin was explored during spontaneous differentiation. During previous spontaneous differentiation studies, the higher levels of vimentin expression correlated with higher levels of mesodermal expression in ipscs compared to ESCs. In addition, force-mediated differentiation caused increased vimentin expression in ESCs. Therefore, vimentin knockout mescs were analyzed and compared to ESCs both in an AggreWell EB model. AggreWells were initially used to aggregate the VIM KO ESCs into EBs because the cells were not forming EBs in the standard rotary EB model. VIM KO-EBs and ESC-EBs both formed rounded cell clusters after one day of culture in AggreWells (Figure 3.12A and Figure 3.13A, respectively). Cells within the ESC-EBs proliferated, as observed in the increase in EB size between day 1 and day 8 (Figure 3.13). Conversely, VIM KO-EBs did not have as high a level of proliferation, as seen in the similar sizes of EBs over the culture duration (Figure 3.12). Vimentin expression was analyzed to verify that these cells were indeed vimentin deficient. ESC-EBs started with low levels of VIM expression which then increased in expression, while VIM KO-EBs maintained low levels of VIM expression throughout 4-6 days of differentiation (Figure 3.14A), validating that these cells were indeed vimentin knockout cells. Vimentin is highly expressed in cells of the mesoderm lineage. Therefore, Mesodermal markers T-BRACHY and FLK1 were then evaluated to determine if

47 38 vimentin had an effect on the cells mesodermal differentiation capabilities. T-BRACHY expression increased for ESCs-EBs and then continuously decreased during the differentiation period, a typical expression profile for this transient marker. VIM KO-EB levels, conversely, remained consistently low (Figure 3.14B). For FLK1, ESC-EBs increased in expression, while VIM KO-EBs had very low expression levels for the entire duration of differentiation (Figure 3.14C). Thus, VIM KO-EBs had lower mesodermal gene expression levels than that of ESC-EBs, indicating that vimentin may play an important role in mesodermal differentiation.

48 39 Figure Phase Images of AggreWell VIM KO ESC-EBs. VIM KO ESCs were differentiated as EBs in AggreWells for four days and then allowed to continue differentiation as EBs in static conditions for four days. Scale bar represents 200µm.

49 40 Figure Phase Images of AggreWell ESC-EBs. ESCs were differentiated as EBs in AggreWells for 24 hours and then continued differentiation as EBs in rotary conditions for seven days. Scale bar represents 200µm.

50 41 Figure Gene expression of ESCs and VIM KO ESCs as EBs. ESCs were differentiated as EBs and assessed for gene expression at day 0, 1, 2, 3, 4, 5, 6, 7, and 8 and VIM KO ESCs were differentiated as EBs and assessed for gene expression at day 0, 2, 3, 4, 6, and 7. Genes analyzed were VIM, T-BRACHY and FLK1. Data presented are averages (mean ± SEM, n = total of 3 replicates from 3 independent trials) for ESC EBs, while VIM KO ESC EBs and day 0 samples have n =1.

51 42 4. DISCUSSION In these in vitro studies, ESCs and ipscs were differentiated as EBs and exposed to applied fluid shear stress to quantify cytoskeletal gene expression during spontaneous and directed differentiation. We first developed a model of differentiation for ipscs comparable to an established ESC differentiation model. After 8 days of spontaneous differentiation, ipsc-ebs had higher mesodermal and cytoskeletal gene expression levels compared to ESC-EBs. Force-mediated differentiation, however, resulted in increased mesodermal gene expression for both ESCs and ipscs, but induced different cytoskeletal responses between the two phenotypes. The different mesodermal and cytoskeletal responses in ipscs and ESCs prompted further investigation into the cytoskeleton s roles during differentiation. Therefore, the role of the intermediate filament vimentin was explored during spontaneous differentiation using vimentin knockout ESCs. VIM KO- EBs had lower mesodermal gene expression levels than that of ESC-EBs, indicating that vimentin may play an important role in mesodermal differentiation. Overall, these studies indicate that cytoskeletal remodeling of ipscs differs from that of ESCs during differentiation, which may be due to residual properties from the fibroblastic parental cell source of ipscs. In addition, the cytoskeleton may play a role in differentiation. Studies have shown the effects of ipscs derived from different somatic cell origins, with differences in gene expression patterns [26] and functionality [6, 27] of the cell lines. These differences ultimately affect their in vitro differentiation capabilities [8, 28, 29]. Reprogramming (de-differentiating) somatic cells back into a stem cell state (ipscs) is associated with a loss in the extensive cytoskeleton. However, ipscs may have higher initial expression levels than ESCs at the stem cell state. Re-differentiation then

52 43 causes the reformation of the cytoskeleton (Figure 4.1). The higher levels of cytoskeletal expression in ipscs during differentiation may result from the previous existence of a cytoskeleton, whereas ESCs have not yet had a well-developed cytoskeleton. Previously, our lab has assessed the cytoskeletal state of ipscs, their fibroblastic parental source, and ESCs. These studies found that ipscs had higher cytoskeletal expression levels than ESCs but significantly lower expression levels than their parental MEFs. The higher cytoskeletal expression levels in ipscs indicated residual properties, possibly due to incomplete reprogramming. For example, a cell may not be completely reprogrammed to the pluripotent state or only a fraction of the cells in the population may be reprogrammed [8, 9]. The EB differentiation model, which is based on only the pluripotent stem cells, eliminates any contaminating non-pluripotent cells. Thus, the results of accelerated and increased expression of mesodermal and cytoskeletal markers in EBs indicate a greater propensity of reprogrammed to cells to re-differentiate to the lineage of their parental phenotype.

53 44 Figure 4.1. A possible model of de-differentiation and re-differentiation of PSCs. Fibroblasts are reprogrammed into a stem cell-like state as ipscs (de-differentiation) and then allowed to re-differentiate. During de-differentiation, fibroblasts lose cytoskeletal and mesodermal expression until they reach the ipsc state, which has higher initial expression than that of ESCs. Then, upon re-differentiation, ipscs regain cytoskeletal and mesodermal expression at a faster rate than ESCs.

54 45 ipscs generated from different parent cell types retain epigenetic memory. For example, in differentiation toward bone, fibroblast-derived ipscs produced more cells expressing osteogenic genes than blood-derived ipscs. Conversely, in differentiation toward blood, blood-derived ipscs produced more hematopoietic colonies than fibroblast-derived ipscs [8]. Blood and bone cells have different cytoskeletons. These differentiation patterns could be due, in part, to the residual cytoskeleton. The same pattern was also seen in neonatal keratinocytes (NK) and umbilical cord blood cells. The NK-iPSCs developed keratin 14 expression at a faster rate compared to blood-derived ipscs. Both cell types were human and from the same patient [30]. Therefore, therapies using ipscs should consider the parental cell source, due to residual properties including the cytoskeletal state and mechanoresponse. Early passage ipscs have been shown to retain transcriptional and epigenetic memory of their parental cell source, losing these molecular and functional differences with continued passaging [28]. Complete ipsc reprogramming, therefore, is a gradual process to reach an ESC-equivalent ground state. Related research findings have demonstrated that different early passage ipsc lines had a tendency to differentiate preferentially to the cell lineage of the somatic cell of origin [28, 31]. Our use of Passage 3 ipscs (originally obtained as Passage 0 ipscs) in the differentiation experiments may therefore be complicated by incomplete reprogramming from the fibroblastic state. Fluid shear stress is known as a regulator of directed stem cell differentiation. However, cytoskeletal remodeling with applied shear stress is still unclear. Numerous studies have found similarities, as well as differences, in ipscs and ESCs, which may play a role in their distinct differentiation capabilities. For example, researchers have

55 46 found differences in human ipscs and ESCs in their epigenetic memory [8], protein expression and phosphorylation [32], and mechanical properties [33]. These studies suggest that these disparities seen in ipscs may in part be due to residual properties from the tissue of origin as a result of incomplete reprogramming, as described above. In particular, Daniels et al finds that hipscs were more viscous than hescs, which had some elastic regions, suggesting that changes in visocoelastic properties may correlate with its degree of differentiation [30]. Such differences in cell stiffness may account for our observations in force-mediated differentiation of stem cells. Cell stiffness is shown to depend on the interplay between the cytoskeletal filaments [35]. Our applied shear stress model resulted in higher gene expression levels for ipscs compared to ESCs for all genes tested, supporting the possibility of residual cytoskeletal elements from their fibroblastic parental cell source. Interestingly, the cellular deformations due to fluid flow in our model did not induce changes in the gene expression of the ESC actin network, which is a major contributor to cell size and shape. Intermediate filaments are known to contribute to the cytoskeleton, but their precise functions are still unclear. This bioreactor system, which induces remodeling of these elements in certain phenotypes, may be a useful tool to further explore the functions of intermediate filaments both individually and collectively within a cytoskeletal network. However, this model of differentiation does not account for a heterogeneous population of cells. Both spontaneous and directed differentiation caused different mesodermal and cytoskeletal responses motivating the investigation of the cytoskeleton s role in differentiation. Specifically, vimentin knockout ESCs were tested in an AggreWell EB model to assess the vimentin intermediate filament s involvement in differentiation.

56 47 Vimentin is highly present in cells of mesenchymal origin, such as fibroblasts, and plays a role in cell motility, cell shape, and endurance of mechanical stress of mesenchymal cells [36, 37]. Other studies have found that vimentin-deficient fibroblasts exhibited reduced collagen synthesis [38], which relates to findings of impaired wound healing in vimentin knockout mice [39, 40]. MEFs, which have high vimentin expression levels, are of mesoderm origin, further motivating the mesodermal differentiation analysis. Finding that, upon differentiation, vimentin knockout ESCs expressed lower mesodermal levels than that of ESCs suggests that vimentin may play an important role in mesodermal differentiation. Furthermore, inhibited differentiation potentially could affect cell behavior and function. Further studies are needed with the vimentin knockout ESC EBs. This initial study was of only a single trial. Therefore, repeating the experiment is critical. Furthermore, analyzing each day of differentiation would improve the comparison to the ESC EB model and allow for assessment of vimentin expression levels for all time points. In addition, ESC-EBs cultured in AggreWells for 1 day compared to VIM KO- EBs cultured in AggreWells for 4 days may cause different effects at specific days of differentiation. Even with these limitations, this preliminary vimentin knockout ESC embryoid body experiment provides insight to the potential role of vimentin in mesodermal differentiation. Future directions may involve investigating the potential role of the cytoskeleton during the process of somatic cell reprogramming. For example, including cytoskeletonmodifying drugs into protocols could change reprogramming efficiency. Picking or sorting for fully reprogrammed cells would also help remove some heterogeneity.

57 48 Assessments on the single cell level, such as flow cytometry analysis or cell stiffness and shape analysis using atomic force microscopy (AFM), could account for any potential effects due to reprogramming inefficiency of ipscs. Extensive studies using other cytoskeletal deficient cells could provide a better understanding on how the cytoskeleton as a whole affects differentiation.

58 49 6. CONCLUSION The overall objective of this study was to determine gene expression of cytoskeletal elements in PSCs during differentiation as well as identify the role of the cytoskeleton in differentiation. Our overarching hypothesis was that residual properties from the parental fibroblast cell source of ipscs would alter cytoskeletal expression and mesodermal differentiation of ipscs compared to ESCs. Further, we hypothesized that the differences in mesodermal differentiation were the result of cytoskeletal differences indicating that the cytoskeleton plays a role in differentiation. Spontaneous differentiation and applied fluid shear stress both serve as robust models for the analysis of cytoskeletal remodeling during differentiation. In both differentiation models, ipscs tended to differentiate toward the mesoderm lineage and had higher cytoskeletal expression than ESCs, which may, in part, be due to residual properties from the original fibroblastic parental cell source. ipscs were found to have differences in cytoskeletal remodeling and differentiation capabilities compared to ESCs, motivating the analysis of the cytoskeleton s function in differentiation. In particular, vimentin knockout ESC studies gave insight into the important role vimentin plays in mesodermal differentiation. Consequently, therapies based on ipscs may need to take into account residual properties from the parental cell source, such as the cytoskeletal state and the mechanoresponse. Personalized medicine approaches may thus strategically select cell sources for reprogramming based on the mechanical microenvironment of the in vivo transplantation site. Taken together, these studies highlight the importance of the cell state of ipscs after reprogramming. Furthermore, they highlight the need for improved understanding of the

59 50 cytoskeleton during differentiation. This approach will help realize the potential of ipscs as cell sources for personalized medicine.

60 51 APPENDIX APPENDIX A: ipsc EXPANSION TRIALS Preliminary expansions of ipscs involved expanding Passage 1 ipsc vials. 1 vial, 1/2 vial, and 1/4 vial of Passage 1 ipscs were cultured for up to six days and analyzed using flow cytometry for protein analysis. These initial studies showed a low number of dome-like highly refractive colonies (Figure A.1). However, cells from the 1/2 vial expansion had similar morphology with rounded colonies and refractive edges compared to ESCs (Figure 3.2). A subpopulation of cells for the 1/2 vial expansion had similar increased pluripotency (NANOG) protein expression compared to ESCs (Figure A.2). For initial experiments, 1/2 vial of Passage 1 ipscs was chosen due to morphology and pluripotency protein expression similar to that of ESCs.

61 52 Figure A.1. Morphology of Passage 1 ipscs. Phase images of 1 vial, 1/2 vial, and 1/4 vial of Passage 1 ipscs after six days of culture on gelatin-coated flasks.

62 53 Figure A.2. Pluripotency expression of Passage 1 ipscs. NANOG protein expression given by histograms of 1 vial, 1/2 vial, and 1/4 vial of Passage 1 ipscs (red lines) compared to ESCs (black lines). BECs (gray line) serve as control.

63 54 APPENDIX B: ENDODERMAL GENE EXPRESSION OF ESCS AND IPSCS AS EBS Using the standard EB model, AFP and SOX17 were analyzed for endodermal expression for up to 8 days of differentiation. SOX17 expression steadily increased in expression level for ESC-EBs, while ipsc-eb expression stayed consistently low, with ESC-EB expression significantly (p<0.001) higher than that of ipsc-ebs at day 8 (Figure B.1). ipsc-ebs and ESC-EBs had similar trends in AFP expression during early differentiation, increasing in expression from day 4 to day 8 (Figure B.1).

64 55 Figure B.1. Endoderm gene expression of ESCs and ipscs as EBs. ESCs and ipscs were differentiated as EBs and assessed for gene expression at day 0, 2, 4, and 12 for endoderm markers (AFP and SOX17). Data presented are averages (mean ± SEM, n = total of 3 replicates from 1 trial), while day 0 samples have n = 1. Significant differences between ESC-EB and ipsc-eb samples at day 8 are indicated by asterisks (*** p<0.001).

65 56 APPENDIX C: Gene Expression of Day 12 ESC-EBs and ipsc-ebs Using our standard EB model, ESC-EBs and ipsc-ebs were assessed at day 12 of differentiation. Pluripotency (Figure C.1), germ lineage (Figure C.1), and cytoskeletal (Figure C.2) gene expression were analyzed at this time point to compare the two phenotypes. Both cell types at day 12 of spontaneous differentiation showed unclear trends compared to other time points tested. This data was from a single trial, therefore, replicating this experiment would allow for a clearer understanding of differentiation at day 12.

66 57 Figure C.1. Pluripotency and germ lineage gene expression of ESCs and ipscs as EBs. ESCs and ipscs were differentiated as EBs and assessed for gene expression at day 12 for a pluripotency marker (NANOG), mesoderm markers (T-BRACHY and FLK1), and endoderm markers (AFP and SOX17). Data presented are averages (mean ± SEM, n = total of 3 replicates from 1 trial).

67 58 Figure C.2. Cytoskeletal gene expression of ESCs and ipscs as EBs. ESCs and ipscs were differentiated as EBs and assessed for gene expression at day 12 for intermediate filaments (VIM, LMNA, KRT8, and NES), microtubules (TUBA1B), and microfilaments (ACTA2). Data presented are averages (mean ± SEM, n = total of 3 replicates from 1 trial).