Poster L17 - Evaluating a Novel Cell Source for Tissue Regeneration

Thursday, October 24, 2024

2:45 PM – 3:45 PM EST

Location: Exhibit Hall E, F & G

Presenting Author(s)

Vidhika Siddha

Student
Northwestern University
Evanston, Illinois, United States

Co-Author(s)

MG

Madeleine Goedegebuure, MS

Graduate Student
Northwestern University
Evanston, Illinois, United States
MB

Matthew Bury

Lab Manager
Lurie Children's Hospital, United States

Primary Investigator(s)

GA

Guillermo Ameer

Daniel Hale Williams Professor of Biomedical Engineering, McCormick School of Engineering Professor
1 Department of Biomedical Engineering, Northwestern University, 2 Center for Advanced Regenerative Engineering (CARE), 3 Department of Surgery, Northwestern University Feinberg School of Medicine, United States

Last Author(s)

AS

Arun Sharma, PhD

Professor
Northwestern University, United States

Introduction: Patients with severe bladder diseases or trauma face chronic bladder dysfunction. Currently, the last-resort treatment option is surgical reconstruction of the bladder, which is invasive and high risk, because it typically involves grafting bowel tissue to the bladder. Cell-seeded biomaterial scaffolds have emerged as alternative grafts for bladder augmentation. Scaffolds seeded with bone marrow-derived mesenchymal stem cells (bMSCs) and hematopoietic stem cells (bHSPCs) have been shown to successfully regenerate bladder tissue (1-3). However, use of these cells significantly restricts clinical translation of treatments. Namely, cells derived from bone marrow have a high degree of heterogeneity due to patient age and any comorbidities, limited growth ex vivo, and involve invasive isolation, which severely affects consistency and efficacy of cell-based therapeutics (4, 5). Alternatively, iPSCs derived from skin fibroblasts can be differentiated into MSCs and HSPCs (iMSCs and iHSPCs, respectively). iMSCs and iHSPCs have emerged as an alternative cell source that maintain the efficacy of bMSCs and bHSCPs while mitigating the previously described limitations. The aim of this work is to compare the regenerative capacity of iMSCs/iHSPCs and bMSCs/bHSPCs in an established bladder regeneration model with the goal of establishing iMSCs/iHSPCs as a viable, alternative cell source for bladder tissue regeneration with greater potential for clinical translation (6, 7). The novel use of iMSCs/iHSPCs for bladder tissue engineering has the potential to change the course of future work in development of alternative treatments for patients with severe and end-stage bladder diseases or injuries.

Materials and

Methods: Both cell sources were evaluated in a previously established rat bladder augmentation model. Poly (octanediol octanol citrate) (POCO) scaffolds were prepared as previously described with a molar ratio of 0.2:0.8:1 (octanol: octanediol: citric acid) and cured at 80°C for 3-4 days. POCO scaffolds were incubated in MSC growth medium for 2 days then seeded with iMSCs or bMSCs at a density of 15,000 cells/cm2. After 7-10 days, the medium was replaced with a 1:1 mixture of MSC growth medium and HSPC growth medium. CD34+ iHSPCs or bHSPCs were seeded on the scaffolds at 100,000 cells/scaffold. After 24 hours, the scaffolds were used for bladder augmentation in athymic nude rats. Prior to surgery and four weeks post-augmentation, urodynamic testing was performed. Bladder capacity, compliance, and void frequency were measured.
After four weeks, the animals were sacrificed and bladder tissue was collected and processed for analysis. Muscle: collagen ratio, blood vessel number and surface area, urothelium thickness, and peripheral nerve length were quantified for each experimental group.
In vitro analysis consists of trilineage differentiation, live/dead staining, and flow cytometry. Using commercially available kits, both MSC populations are differentiated into chondrocytes, adipocytes, and osteocytes, and histological stains are used to demonstrate their differentiation capacity. Live/dead staining of both cell types on POCO scaffolds is conducted to quantify cell viability. Lastly, each cell population is assessed for known positive and negative MSC and HSPC markers using commercially available flow cytometry kits.

Results, Conclusions, and Discussions: Bladder function and regeneration of the bladder wall components were compared pre- and 4 weeks post-augmentation. Urodynamic testing was completed to determine bladder capacity, compliance, and void frequency before and 4 weeks after augmentation. There were no statistically significant differences in these bladder function parameters between the iMSC/iHSPC group and the bMSC/bHSPC group. On average, all groups showed a 30-40% increase in capacity post-augmentation. Bladder compliance and void frequency both remained within a normal range post augmentation for all groups (0.72 mg/cm H2O compliance and 1.3-1.4 voids/min). This indicates that iMSCs/iHSPCs supported normal bladder activity, similar to previously published results with bMSCs/bHSPCs (3).
Histological assessment of the regenerated bladder tissue also revealed that results from iMSC/iHSPC and MSC/HSPC groups had no statistically significant difference. After 4 weeks of augmentation, both groups had muscle:collagen ratios around 0.6 and urothelium thickness of 51µm, values comparable to those of native tissue. Furthermore, both groups had comparable blood vessel regeneration and average peripheral nerve length: average blood vessel size was around 300µm with 250 vessels/mm2, and average nerve length was 40.5µm.
In vitro analyses (flow cytometry, trilineage differentiation, and live/dead analysis) will also be conducted to validate the results of in vivo experiments. With both in vivo and in vitro data, it will thus be shown that iPSC-derived MSCs and HSPCs are comparable to their bone marrow-derived counterparts in regenerating bladder tissue.

Acknowledgements (Optional): I would like to thank my mentor and PhD candidate Madeleine Goedegebuure, Matthew Bury, Dr. Guillermo Ameer, and Dr. Arun Sharma for supporting and guiding this project.

References:
1. Sharma, A. K., Hota, P. V., Matoka, D. J., Fuller, N. J., Jandali, D., Thaker, H., Ameer, G. A., & Cheng, E. Y. (2010). Urinary bladder smooth muscle regeneration utilizing bone marrow derived mesenchymal stem cell seeded elastomeric poly(1,8-octanediol-co-citrate) based thin films. Biomaterials, 31(24), 6207–6217. https://doi.org/10.1016/j.biomaterials.2010.04.054

2. Sharma, A. K., Bury, M. I., Fuller, N. J., Rozkiewicz, D. I., Hota, P. V., Kollhoff, D. M., Webber, M. J., Tapaskar, N., Meisner, J. W., Lariviere, P. J., Destefano, S., Wang, D., Ameer, G. A., & Cheng, E. Y. (2012). Growth factor release from a chemically modified elastomeric poly(1,8-octanediol-co-citrate) thin film promotes angiogenesis in vivo. Journal of biomedical materials research. Part A, 100(3), 561–570. https://doi.org/10.1002/jbm.a.33306

3. Sharma, A. K., Bury, M. I., Fuller, N. J., Marks, A. J., Kollhoff, D. M., Rao, M. V., Hota, P. V., Matoka, D. J., Edassery, S. L., Thaker, H., Sarwark, J. F., Janicki, J. A., Ameer, G. A., & Cheng, E. Y. (2013). Cotransplantation with specific populations of spina bifida bone marrow stem/progenitor cells enhances urinary bladder regeneration. Proceedings of the National Academy of Sciences of the United States of America, 110(10), 4003–4008. https://doi.org/10.1073/pnas.1220764110

4. Demirci, S., Leonard, A., & Tisdale, J. F. (2020). Hematopoietic stem cells from pluripotent stem cells: Clinical potential, challenges, and future perspectives. Stem Cells Translational Medicine, 9(12), 1549–1557. https://doi.org/10.1002/sctm.20-0247

5. Siegel, G., Kluba, T., Hermanutz-Klein, U., Bieback, K., Northoff, H., & Schäfer, R. (2013). Phenotype, donor age and gender affect function of human bone marrow-derived mesenchymal stromal cells. BMC Medicine, 11(1). https://doi.org/10.1186/1741-7015-11-146

6. Lee, H.-R., Kim, S., Shin, S., Jeong, S.-Y., Lee, D.-W., Lim, S.-U., Kang, J. Y., Son, M.-Y., Lee, C., Yu, K.-R., Kim, M., & Oh, I.-H. (2023). iPSC-derived MSCs are a distinct entity of MSCs with higher therapeutic potential than their donor-matched parental MSCs. International Journal of Molecular Sciences, 24(1), 881. https://doi.org/10.3390/ijms24010881

7. Netsrithong, R., Suwanpitak, S., Boonkaew, B., Trakarnsanga, K., Chang, L. J., Tipgomut, C., Vatanashevanopakorn, C., Pattanapanyasat, K., & Wattanapanitch, M. (2020). Multilineage differentiation potential of hematoendothelial progenitors derived from human induced pluripotent stem cells. Stem cell research & therapy, 11(1), 481. https://doi.org/10.1186/s13287-020-01997-w