Human Induced Pluripotent Stem Cell Derived Neural Stem Cell

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Letter

Human induced pluripotent stem cell derived neural stem cell survival and neural differentiation on polyethylene glycol dimethacrylate hydrogels containing a continuous concentration gradient of n-cadherin derived peptide His-Ala-Val-Asp-Lle Hyun Ju Lim, Zara Khan, Thomas Wilems, Xi Lu, T. Hiran Perera, Yuki Ellen Kurosu, Krishna Teja Ravivarapu, Matthew C. Mosley, and Laura A. Smith Callahan ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.6b00745 • Publication Date (Web): 14 Apr 2017 Downloaded from http://pubs.acs.org on April 18, 2017

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ACS Biomaterials Science & Engineering

Human induced pluripotent stem cell derived neural stem cell survival and neural differentiation on polyethylene glycol dimethacrylate hydrogels containing a continuous concentration gradient of n-cadherin derived peptide His-Ala-Val-AspLle. Hyun Ju Lim†‡, Zara Khan†‡, Thomas S. Wilems†, Xi Lu†, T. Hiran Perera†, Yuki E. Kurosu†, Krishna T. Ravivarapu†, Matthew C. Mosley†, and Laura A. Smith Callahan*†§. AUTHOR ADDRESS. † The Vivian L. Smith Department of Neurosurgery and Center for Stem Cells and Regenerative Medicine, McGovern Medical School at the University of Texas Health Science Center at Houston. 1825 Pressler, Houston, Texas 77030. and § Department of Nanomedicine and Biomedical Engineering. McGovern Medical School at the University of Texas Health Science Center at Houston. 1825 Pressler, Houston, Texas 77030. E-mail: [email protected]. Tel.: 1-713-3431. Fax: 1-713-500-2424.

ABSTRACT: Although preclinical models of spinal cord injury have shown that matrix inclusion in stem cell therapy leads to greater neurological improvements than including cells alone, there has been insufficient matrix optimization for human cells. Ncadherin influences the development and maintenance of neural tissue, but the effects of n-cadherin derived peptide His-Ala-ValAsp-Lle (HAVDI) on the survival, neurite extension and expression of neural differentiation markers in human induced pluripotent stem cell derived neural stems (hNSC) have not been widely examined. Using polyethylene glycol hydrogels containing a continuous gradient of HAVDI, this study identifies concentration dependent effects on hNSC survival and neural differentiation. KEYWORDs. Human Pluripotent Stem Cells, Neural, N-Cadherin, Tissue Engineering, and Combinatory Approach Stem cell therapy offers patients suffering from spinal cord injury the hope of restoring lost neurological function that is not offered by current clinical treatments1. Typically, isolated stem cells are injected directly into the lesion site, which is unsupportive of cellular survival and engraftment. This experimental treatment protocol has demonstrated only modest improvements in neurological function

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. However, preclinical data indicate that inclusion of a biomaterial support for the cells can improve the restoration of

neurological function beyond that achieved with cellular injection alone2, 4. The inclusion of advanced biomaterial supports with optimized properties and bioactive signaling may lead to even greater improvements in neurological function. Although the development of biomaterials for neural tissue engineering is a rapidly evolving field, little work has been done to optimize the matrices for clinically relevant human cell types2, 4-6.

As culture of these cell types is expensive and difficult, use of combinatory methods,

such as arrays and continuous gradients, to expedite the identification of promising biomaterials and tethered signaling conditions will facilitate the progression of combination cell/biomaterial treatments to the clinic7. N-cadherin (NCAD) plays an important but complex role in the development and homeostasis of the central nervous system (CNS)8. As such, cellular response to NCAD likely depends on NCAD concentration, cellular maturation state and cell type9-10.

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Several studies investigating emulated NCAD on biomaterials have shown mixed results in achieving the desired cellular response

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in neural regenerative applications11-15. In addition, only a single study utilized a clinically relevant human neural cell type5. Cellular response to biomaterials can vary based on the maturation state and species of the cells16-17. To understand how biomaterials will affect stem cell therapy treatment outcomes, more testing with clinically relevant neural cell types, such as human induced pluripotent stem cell derived neural stem cells (hNSC), should be conducted. hNSC are of particular interest because they eliminate ethical concerns and medical complications due to long term immunosuppression that surround other cell sources, such as human embryonic stem cells (hESC) and fetal tissue derived cells. Most of the previous studies utilize Fc-tagged recombinant NCAD5, 11-13. However, a NCAD derived peptide His-Ala-Val-Asp-Lle (HAVDI) may be a superior candidate for inclusion into a biomaterial as it is easier to evenly disperse through matrices due to its smaller size. The peptide can stimulate similar cellular receptors and pathways compared to native NCAD18-19. Given the lack of investigation in the literature, cellular response to HAVDI should be studied further for neural applications in order to determine if it provides similar stimulation of cellular receptors and can be used interchangeably with other emulated NCAD. Previous studies examining the effects of NCAD concentration on cellular response have used only a limited number of discrete samples5, 14, which may misidentify the optimal signaling conditions. The present study utilizes a combinatory approach to optimize HAVDI concentration for maximal hNSC survival, neurite extension and gene expression of neural differentiation markers. Although many combinatory approaches have been developed7, a continuous gradient approach was selected to maximize the number of tested HAVDI concentrations while minimizing the number of hNSC needed to conduct the studies. Due to its low protein absorption and limited cellular adhesion20, polyethylene glycol dimethacrylate (PEG) was selected as the base polymer to study the effects of HAVDI concentration on hNSC survival and neural differentiation in order to maximize the effects of peptide-cell interactions on cellular behavior by limiting other types of cell-matrix interaction (Figure 1). A cysteine attached to a three glycine spacer was added to the c-terminus of the n-terminus acetylated HAVDI peptide to allow covalent tethering into the hydrogel. Using a computer controlled syringe pump system (Figure 1B), PEG hydrogels containing a continuous gradient of HAVDI were fabricated and characterized similarly to previously described (further experimental details are available in the supporting information)21. The HAVDI tethered hydrogels were found to contain a linear concentration gradient of HAVDI along the length of the hydrogel using a Lowry assay (Figure 1E). No significant variation of other material properties was observed (Figure 1F- further experimental details are available in the supporting information). Disks were then punched out down the length of the gradient hydrogel (Figure 1D) and placed in 48 well tissue culture plates. An average HAVDI concentration decrease of 39 µM ± 12 µM (reported as mean ± standard deviation across the gradient) was measured across a 5mm span in the center of the punched disks regardless of the gradient position from which the disk originated (further experimental details are available in the supporting information). Segmentation of the gradient was necessary to facilitate cellular attachment in two dimensional culture and limit the effects of crosstalk between cells exposed to significantly different HAVDI concentration ranges.


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Since clinical stem cell therapy treatments will require expansion of the progenitor cell population in order to obtain enough cells

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for treatment, ND2.0 human induced pluripotent stem cells were differentiated for 10 days according to a published protocol22. The derived neural stem cells (NSC) were then expanded on Matrigel coated tissue culture plastic in serum free media. For biological tests, 200,000 hNSC (expansion passage 15) were then seeded on each disk from the HAVDI gradient hydrogel (see supporting information for additional details). Presentation of NCAD to cells reduces apoptosis resulting from multiple types of insult23-24. To mimic the oxidative stress experienced by hNSC injected into spinal cord injury lesions, hNSC were allowed to attach to HAVDI concentration gradient hydrogels and were then exposed to various concentrations of hydrogen peroxide (H2O2) for 24 hrs. After which, cellular viability was determined using an MTS assay (Figure 2). HAVDI concentrations of 577µM and above in the hydrogel were found to increase hNSC survival compared to hNSC cultured on lower concentration HAVDI PEG hydrogels or Matrigel coated tissue culture plastic when 310µM of H2O2 was added to the culture. When higher H2O2 concentration exposures were used the inclusion of HAVDI in the hydrogel was found to either promote cellular survival regardless of concentration (980µM H2O2) or provide no benefit to cellular survival compared to Matrigel coated tissue culture plastic (620µM & 1860µM H2O2). The higher H2O2 concentrations could lead to greater reductions NCAD at the cellular membrane and the inhibition of downstream signaling pathway activation 25-26. The ability of HAVDI to bind the remaining hNSC receptors could have been affected due to conformation changes in the peptide or extracellular receptor domains due to PH or oxidation changes in the culture that altered salt bridges, ionic interactions and hydrogen bonds necessary for receptor binding and stimulation23, 26-27. H2O2 degradation of HAVDI is unlikely in this study because a previous study reported HAVDI concentration dependent survival of mouse embryonic stem cells at higher H2O2 concentrations with lower HAVDI concentrations in PEG hydrogels15. In contrast to the observed results, a study of hESC derived NSC found survival to decrease after 6 hrs of exposure to 20 µM H2O2 with increasing Fc-tagged recombinant NCAD concentration in the matrix5. Although experimental design differences likely play a role, the discrepancy in response between these studies indicate that the response to NCAD may depend on the type of the cells, the maturation state of the cells, or variation in receptor stimulation by HAVDI and Fc-tagged recombinant NCAD. Neural differentiation and neurite extension are affected by the concentration of emulated NCAD presented to cells5, 14-15. After 10 days of neural differentiation on the HAVDI concentration gradient hydrogels, hNSC were stained for β III tubulin (TUJ1) (Figure 3A). Significantly more hNSC cultured on the 779 µM HAVDI hydrogels stained positively for TUJ1 compared to hNSC cultured on hydrogels containing 578 µM of HAVDI (Figure 3B). The percentage of hNSC expressing neurites was not affected by HAVDI concentration (51.7% ± 11.3%, reported as mean ± standard deviation across the gradient); these results are similar to a previous study of mESC response to culture on HAVDI containing hydrogels15. However, a study with hESC derived NSC found that increasing NCAD concentration decreased the percentage of cells expressing neurites and the average neurite length on poly(DTEco-10% DT-co-10% PEG1k) films and electrospun fibers5. The present study showed a similar reduction in neurite length in hNSC cultured on higher HAVDI concentration PEG hydrogels (Figure 3C). Studies of mESC and embryonic chicken dorsal root ganglia


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identified a biphasic response in average neurite length to different concentrations of NCAD peptides on PEG and fibrin hydrogels,

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respectively14-15. Although the studies with human neural cell types may not have included low enough concentration of the peptide to identify a biphasic response in neurite extension, it is more likely that these differences in cell response are based on species or maturation state. NCAD signaling is known to transmit mechanical stiffness from the matrix to the cells 28, which affects cellular differentiation and neurite length6, 29-30. Additionally, changes in the mechanical stiffness of the matrix can alter the expression of NCAD on the cells31, which can affect growth cone velocity leading to further changes in cellular polarization and neurite length32. Therefore, differences in matrix stiffness likely played a significant role in the observed differences between studies. The current study found a concentration dependent effect on mRNA gene expression of neural differentiation markers (primers and conditions are available in the supporting information and Table S1) in hNSC cultured on hydrogels containing a continuous gradient of HAVDI (Figure 4). Early neural stem cell marker, SRY-box 1 (Sox1), exhibited the lowest expression in hNSC cultured on hydrogels containing 577µM of HAVDI after 10 days of neural differentiation (Figure 4A), while neuronal markers TUJ1 (Figure 4B) and microtubule associated protein 2 (MAP2) (Figure 4C) exhibited maximal expression in hNSC cultured on hydrogels containing 577 µM of HAVDI. It is not uncommon for the mRNA expression of a protein and the amount of protein in cells to be dissimilar33. As observed in the current study, maximal mRNA expression of TUJ1 occurs in hNSC cultured on the same HAVDI concentration hydrogels where the lowest percentage of cells stained positively for TUJ1 by immunofluorescence. Since gene expression is a population average measurement, the higher TUJ1 gene expression could result from greater neuronal maturation in the TUJ1 positive cells cultured on the 577 HAVDI concentration hydrogels compared to hNSC cultured on hydrogels containing other HAVDI concentrations. The reduced percentage of cells staining positive for TUJ1 at this HAVDI concentration could be indicative of increased cellular commitment to other neural lineages, such as oligodendrocytes or astrocytes. Although the concentration range of maximal TUJ1 mRNA expression in the hNSC is shifted toward higher concentrations of HAVDI, it does overlap with the range identified in a previous study of mESC response to HAVDI concentration15. Another study with murine cells cultured on NCAD coated surfaces identified a similar reduction of stem cell markers and increased expression neuronal markers with exposure to NCAD compared to the control culture surface34. Exploiting the ability of NCAD signaling to promote neural differentiation in immature cells could serve as a last line safety measure to inhibit teratoma formation from under differentiated cells in the implantation treatment population for stem cell therapy that were not removed by other quality control measures. Maximal oligodendrocyte transcription factor 2 (Olig2) mRNA gene expression was observed in hNSC cultured on 577 µM HAVDI hydrogels. (Figure 4D). Olig2 expression can be an indicator of both motor neuron and oligodendrocyte progenitor differentiation. However, when coupled with decreased cellular staining for TUJ1 in hNSC cultured on the 577 µM HAVDI hydrogels compared to hNSC cultured on 760 µM HAVDI hydrogels (Figure 3B), the observed Olig2 expression is more likely to denote the presence of oligodendrocyte progenitors than motor neurons. Oligodendrocytes would facilitate myelination of new axon exten-


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sions from implanted hNSC and host neurons in addition to remyelination of intact axons in the CNS lesion. Inclusion of a signal-

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ing peptide, which can increase axon extension and myelination, would be beneficial for functional recovery after SCI. A previous study of mESC and mouse induced pluripotent stem cell differentiation showed a promotion of neuronal differentiation over astrocyte differentiation on NCAD tethered surfaces 13. This trend was not observed in the present study and HAVDI concentration was found to have no effect on the mRNA expression of glial fibrillary acidic protein (GFAP) by hNSC (Figure 4E). A number of inconsistences between the current study using HAVDI and previous studies using alternative NCAD emulation, such as with Fc-tagged recombinant NCAD, have been identified. The inconsistences could due to differences in the how HAVDI and other emulated NCAD bind and stimulate cellular receptors, which would indicate that they may not be fully interchangeable. Differences in the maturation state and types of cells utilized in the studies could also play a role. Future studies directly comparing multiple types of NCAD emulation on cellular response will be necessary to resolve these issues. However, the effects of the different tethering chemistries on matrix properties, which could significantly affect cellular response, need to be addressed before these studies can be conducted. NCAD signaling is a promising candidate for inclusion in tethered bioactive signaling schemes in regenerative matrices to support stem cell therapy. Unlike a significant portion of the literature utilizing gradient materials as chemoattractant/repellents35-37, the current work utilizes a concentration gradient hydrogel to identify a HAVDI concentration of 577µM tethered in PEG hydrogels as optimal for improvement of hNSC survival after exposure to oxidative stress, and promoting neurite extension and maturation toward neural lineages. All of which, are important characteristics for matrices included in stem cell therapy protocols. Inclusion of the current optimized HAVDI matrix in stem cell therapy protocols would likely lead to improvements in the restoration of neurological function beyond what is achieved by the implantation of cells alone. However, NCAD is just one part of the native bioactive signaling that occurs during neural tissue development. Presentation of multiple peptides to stimulate synergistic signaling pathways, such as NCAD and integrin, in matrices will likely lead to further improvements in restoration of neurological function from matrix supported stem cell therapy in SCI38. As crosstalk between stimulated pathways could alter cellular response to the matrix and the optimal concentration of bioactive signaling moieties39-42, systematic approaches, such as the one utilized in the current study, should be used to efficiently address the potential need for re-optimization of the presented bioactive signaling moiety concentrations. Further work developing matrices capable of presenting elements of these tethered bioactive signaling cocktails at independent concentrations needs to be done, as currently only a few such matrices exist43-44. In summary, the current study demonstrates that NCAD derived peptide HAVDI has concentration dependent effects on hNSC survival, neurite extension and neural differentiation that were easily identified using a continuous concentration gradient hydrogels. Collectively, the results suggest that a HAVDI concentration of approximately 577µM in matrices is optimal for the survival and neural maturation of hNSC. Optimized bioactive signaling could lead to greater recovery of neurological function beyond that of simple scaffold inclusion in stem cell therapy treatments for spinal cord injury and should be examined in greater detail.


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ASSOCIATED CONTENT AUTHOR INFORMATION Corresponding Author * e-mail: [email protected]

Author Contributions ‡ These authors contributed equally to the work. HL, ZK and LASC conceived the idea and designed the research. HL, ZK, and TSW conducted the cell experiments. ZK, THP, XL, and KTR conducted the material characterization. TSW, YK, MCM and LASC analyzed the biological data. HL and LASC wrote the manuscript. All authors have given approval to the final version of the manuscript.

Funding Sources The present work was supported in part by the following funding sources to LASC: start-up funds from the Vivian L. Smith Department of Neurosurgery William Stamps Farish Foundation Fund; the Memorial Hermann Foundation Staman Ogilvie Fund; and Mission Connect, a TIRR foundation program (grant number: #014-120).

Supporting Information The Supporting Information is available free of charge on the ACS Publications website. Detailed Experimental Methods and PCR Primers (Table S1) (PDF)

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Ganz, A.; Lambert, M.; Saez, A.; Silberzan, P.; Buguin, A.; Mège, R. M.; Ladoux, B., Traction forces exerted through N-cadherin contacts. Biology of the Cell 2006, 98 (12), 721-730. Musah, S.; Wrighton, P. J.; Zaltsman, Y.; Zhong, X.; Zorn, S.; Parlato, M. B.; Hsiao, C.; Palecek, S. P.; Chang, Q.; Murphy, W. L.; Kiessling, L. L., Substratum-induced differentiation of human pluripotent stem cells reveals the coactivator YAP is a potent regulator of neuronal specification. Proceedings of the National Academy of Sciences 2014, 111 (38), 13805-13810. Leipzig, N. D.; Shoichet, M. S., The effect of substrate stiffness on adult neural stem cell behavior. Biomaterials 2009, 30 (36), 6867-6878. Gu, Y.; Ji, Y.; Zhao, Y.; Liu, Y.; Ding, F.; Gu, X.; Yang, Y., The influence of substrate stiffness on the behavior and functions of Schwann cells in culture. Biomaterials 2012, 33 (28), 66726681. Bard, L.; Boscher, C.; Lambert, M.; Mège, R.-M.; Choquet, D.; Thoumine, O., A Molecular Clutch between the Actin Flow and N-Cadherin Adhesions Drives Growth Cone Migration. The Journal of Neuroscience 2008, 28 (23), 5879-5890. Gry, M.; Rimini, R.; Strömberg, S.; Asplund, A.; Pontén, F.; Uhlén, M.; Nilsson, P., Correlations between RNA and protein expression profiles in 23 human cell lines. BMC Genomics 2009, 10 (1), 365. Haque, A.; Adnan, N.; Motazedian, A.; Akter, F.; Hossain, S.; Kutsuzawa, K.; Nag, K.; Kobatake, E.; Akaike, T., An Engineered N-Cadherin Substrate for Differentiation, Survival, and Selection of Pluripotent Stem Cell-Derived Neural Progenitors. PLOS ONE 2015, 10 (8), e0135170. Kapur, T. A.; Shoichet, M. S., Immobilized concentration gradients of nerve growth factor guide neurite outgrowth. Journal of Biomedical Materials Research Part A 2004, 68A (2), 235-243. DeLong, S. A.; Moon, J. J.; West, J. L., Covalently immobilized gradients of bFGF on hydrogel scaffolds for directed cell migration. Biomaterials 2005, 26 (16), 3227-3234. Dodla, M. C.; Bellamkonda, R. V., Anisotropic scaffolds facilitate enhanced neurite extension in vitro. Journal of Biomedical Materials Research Part A 2006, 78A (2), 213-221. Weber, G. F.; Bjerke, M. A.; DeSimone, D. W., Integrins and cadherins join forces to form adhesive networks. Journal of Cell Science 2011, 124 (8), 1183-1193. Cosgrove, B. D.; Mui, K. L.; Driscoll, T. P.; Caliari, S. R.; Mehta, K. D.; Assoian, R. K.; Burdick, J. A.; Mauck, R. L., N-cadherin adhesive interactions modulate matrix mechanosensing and fate commitment of mesenchymal stem cells. Nat Mater 2016, 15 (12), 1297-1306. Le, N. N. T.; Zorn, S.; Schmitt, S. K.; Gopalan, P.; Murphy, W. L., Hydrogel arrays formed via differential wettability patterning enable combinatorial screening of stem cell behavior. Acta Biomaterialia 2016, 34, 93-103. Jung, J. P.; Moyano, J. V.; Collier, J. H., Multifactorial optimization of endothelial cell growth using modular synthetic extracellular matrices. Integrative Biology 2011, 3 (3), 185-196. Lam, J.; Carmichael, S. T.; Lowry, W. E.; Segura, T., Hydrogel Design of Experiments Methodology to Optimize Hydrogel for iPSC-NPC Culture. Advanced Healthcare Materials 2015, 4 (4), 534-539. Zheng, J.; Hua, G.; Yu, J.; Lin, F.; Wade, M. B.; Reneker, D. H.; Becker, M. L., Postelectrospinning "triclick" functionalization of degradable polymer nanofibers. ACS Macro Letters 2015, 4 (2), 207-213. Lim, H. J.; Perera, T. H.; Wilems, T. S.; Ghosh, S.; Zheng, Y. Y.; Azhdarinia, A.; Cao, Q.; Smith Callahan, L. A., Response to di-functionalized hyaluronic acid with orthogonal chemistry grafting at independent modification sites in rodent models of neural differentiation and spinal cord injury. Journal of Materials Chemistry B 2016, 4 (42), 6865-6875.

FIGURE CAPTIONS


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Figure 1. Fabrication and characterization of polyethylene glycol (PEG) hydrogels containing a continuous gradient of n-cadherin

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(NCAD) derived peptide HAVDI. A. Schematic of hydrogel chemistry. B. Schematic of gradient fabrication system. C. Profiles of syringe pump speed during gradient fabrication. D. Picture of gradient hydrogel in custom mold with characterization positions marked along the length of the gradient. E. Lowry Assay quantification of NCAD derived peptide HAVDI content by position down the length of the gradient after 3 days of washing in PBS. F. Characterization for material properties after 24hrs of swelling in PBS. Figure 2. Survival of human induced pluripotent stem cell derived neural stem cells (hNSC) cultured on Matrigel coated tissue culture plastic (TCP) or polyethylene glycol hydrogels containing a continuous concentration gradient of n-cadherin derived peptide HAVDI after a 24 hr expo-sure to oxidative stress from various concentrations of hydrogen peroxide (H2O2). * denotes statistical signifi-cance (p-value less than 0.05) between groups. Figure 3. Expression of β III tubulin (TUJ1) in human induced pluripotent stem cell derived neural stem cells (hNSC) after 10 days of neural differentiation on polyethylene glycol hydrogels containing a continuous gradient of n-cadherin (NCAD) de-rived peptide HAVDI. A. Immunofluorescent TUJ1 staining (green) with nuclear stain (blue). Scale bar =50 µm. B. Quantifi-cation of the fraction of hNSC attached to the NCAD peptide gradient hydrogels staining positive for TUJ1 in immunofluo-rescent images. C. Quantification of neurite length extended from hNSC cultured on the NCAD peptide gradient hydrogels. * denotes statistical significance (p-value less than 0.05) between groups. Figure 4. mRNA expression of neural stem cell and differ-entiation markers in human induced pluripotent stem cell derived neural stem cells (hNSC) over 10 days of neural differentiation on polyethylene glycol hydrogels contain-ing a continuous gradient of ncadherin (NCAD) derived peptide HAVDI. A. SRY-box 1 (Sox1). B. β III tubulin (TUJ1). C. Microtubule associated protein 2 (MAP2). D. Oligodendrocyte transcription factor 2 (Olig2). E. Glial fi-brillary acidic protein (GFAP) * denotes statistical significance (p-value less than 0.05) between groups.


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FIGURES

Figure 1


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Figure 2

Figure 3


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Figure 4


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For Table of Contents Use Only Table of Contents artwork

Human induced pluripotent stem cell derived neural stem cell survival and neural differentiation on polyethylene glycol dimethacrylate hydrogels containing a continuous concentration gradient of n-cadherin derived peptide His-Ala-Val-AspLle. Hyun Ju Lim, Zara Khan, Thomas S. Wilems, Xi Lu, T. Hiran Perera, Yuki Kurosu, Krishna T. Ravivarapu, Matthew C. Mosley, and Laura A. Smith Callahan.


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Figure 1. Fabrication and characterization of polyethylene glycol (PEG) hydrogels containing a continuous gradient of n-cadherin (NCAD) derived peptide HAVDI. A. Schematic of hydrogel chemistry. B. Schematic of gradient fabrication system. C. Profiles of syringe pump speed during gradient fabrication. D. Picture of gradient hydrogel in custom mold with characterization positions marked along the length of the gradient. E. Lowry Assay quantification of NCAD derived peptide HAVDI content by position down the length of the gradient after 3 days of washing in PBS. F. Characterization for material properties after 24hrs of swelling in PBS. 82x82mm (600 x 600 DPI)


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Figure 2. Survival of human induced pluripotent stem cell derived neural stem cells (hNSC) cultured on Matrigel coated tissue culture plastic (TCP) or polyethylene glycol hydrogels containing a continuous concentration gradient of n-cadherin derived peptide HAVDI after a 24 hr exposure to oxidative stress from various concentrations of hydrogen peroxide (H2O2). * denotes statistical signifi-cance (p-value less than 0.05) between groups. 50x30mm (600 x 600 DPI)



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Figure 3. Expression of β III tubulin (TUJ1) in human induced pluripotent stem cell derived neural stem cells (hNSC) after 10 days of neural differentiation on polyethylene glycol hydrogels containing a continuous gradient of n-cadherin (NCAD) de-rived peptide HAVDI. A. Immunofluorescent TUJ1 staining (green) with nuclear stain (blue). Scale bar =50 µm. B. Quantification of the fraction of hNSC attached to the NCAD peptide gradient hydrogels staining positive for TUJ1 in immunofluorescent images. C. Quantification of neurite length extended from hNSC cultured on the NCAD peptide gradient hydrogels. * denotes statistical significance (p-value less than 0.05) between groups. 102x59mm (600 x 600 DPI)


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Figure 4. mRNA expression of neural stem cell and differentiation markers in human induced pluripotent stem cell derived neural stem cells (hNSC) over 10 days of neural differentiation on polyethylene glycol hydrogels containing a continuous gradient of n-cadherin (NCAD) derived peptide HAVDI. A. SRY-box 1 (Sox1). B. β III tubulin (TUJ1). C. Microtubule associated protein 2 (MAP2). D. Oligodendrocyte transcription factor 2 (Olig2). E. Glial fibrillary acidic protein (GFAP) * denotes statistical significance (p-value less than 0.05) between groups 177x214mm (600 x 600 DPI)



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For table of Contents Only Human induced pluripotent stem cell derived neural stem cell survival and neural differentiation on polyethylene glycol dimethacrylate hydrogels containing a continuous concentration gradient of n-cadherin derived peptide His-Ala-Val-Asp-Lle. Hyun Ju Lim, Zara Khan, Thomas S. Wilems, Xi Lu, T. Hiran Perera, Yuki Kurosu, Krishna T. Ra-vivarapu, Matthew C. Mosley, and Laura A. Smith Callahan.

83x47mm (300 x 300 DPI)


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Human Induced Pluripotent Stem Cell Derived Neural Stem Cell

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