Human Mesenchymal Stem Cell Sheets Improve Uterine Incision Repair in a Rodent Hysterotomy Model

Abstract

Objective The study aimed to assess the feasibility of creating and transplanting human umbilical cord mesenchymal stem cell sheets applied to a rat model of hysterotomy, and additionally to determine benefits of human umbilical cord mesenchymal stem cell sheet transplantation in reducing uterine fibrosis and scarring.

Study Design Human umbilical cord mesenchymal stem cell sheets are generated by culturing human umbilical cord mesenchymal stem cells on thermo-responsive cell culture plates. The temperature-sensitive property of these culture dishes facilitates normal cell culture in a thin contiguous layer and allows for reliable recovery of intact stem cell sheets without use of destructive proteolytic enzymes.

We developed a rat hysterotomy model using nude rats. The rat uterus has two distinct horns: one horn provided a control/untreated scarring site, while the second horn was the cell sheet transplantation site.

On day 14 following surgery, complete uteri were harvested and subjected to histologic evaluations of all hysterotomy sites.

Results The stem cell sheet culture process yielded human umbilical cord mesenchymal stem cell sheets with surface area of approximately 1 cm².

Mean myometrial thickness in the cell sheet-transplanted group was 274 μm compared with 191 μm in the control group (p = 0.02). Mean fibrotic surface area in the human umbilical cord mesenchymal stem cell sheet-transplanted group was 95,861 μm² compared with 129,185 μm² in the control group. Compared with control horn sites, cell sheet-transplanted horns exhibited significantly smaller fibrotic-to-normal myometrium ratios (0.18 vs. 0.27, respectively, p = 0.029). Mean number of fibroblasts in cell sheet-transplanted horns was significantly smaller than the control horns (483 vs. 716/mm², respectively, p = 0.001).

Conclusion Human umbilical cord mesenchymal stem cell sheet transplantation is feasible in a rat model of hysterotomy. Furthermore, use of stem cell sheets reduces fibroblast infiltration and uterine scar fibrotic tissue formation during hysterotomy healing, potentially mitigating risks of uterine scar formation.

Key Points

• Stem cell sheet transplanted to hysterotomy promotes myometrial regeneration and reduced fibrotic tissue formation.

• This study demonstrates the feasibility of using human umbilical cord mesenchymal stem cell sheets.

Keywords

cell therapy - cell sheet therapy - scar repair - cesarean section - placenta accrete - uterine rupture - fibrosis - regenerative medicine

Note

T.O. is a founder of CellSeed Inc. which has licenses for the production of certain cell sheet-related technologies and patents from Tokyo Women's Medical University (Japan).

An earlier version of this work was presented at the 2019 Society for Maternal Fetal Medicine Annual Meeting in Las Vegas, NV from February 11 to 16, 2019 (available at: https://www.ajog.org/article/S0002-9378(18)32011-8/pdf).

Publication History

Received: 14 March 2020

Accepted: 04 November 2020

Article published online:
27 December 2020

• References

• 1 Hall MJ, DeFrances CJ, Williams SN, Golosinskiy A, Schwartzman A. National Hospital Discharge Survey: 2007 summary. Natl Health Stat Rep 2010; (29) 1-20 , 24
• 2 Silver RM, Landon MB, Rouse DJ. et al; National Institute of Child Health and Human Development Maternal-Fetal Medicine Units Network. Maternal morbidity associated with multiple repeat cesarean deliveries. Obstet Gynecol 2006; 107 (06) 1226-1232
• 3 Clark EA, Silver RM. Long-term maternal morbidity associated with repeat cesarean delivery. Am J Obstet Gynecol 2011; 205 (6, Suppl): S2-S10
• 4 Boerma T, Ronsmans C, Melesse DY. et al. Global epidemiology of use of and disparities in caesarean sections. Lancet 2018; 392 (10155): 1341-1348
• 5 Martin JA, Hamilton BE, Osterman MJK, Driscoll AK, Drake P. Births: final data for 2017. Natl Vital Stat Rep 2018; 67 (08) 1-50
• 6 Wu S, Kocherginsky M, Hibbard JU. Abnormal placentation: twenty-year analysis. Am J Obstet Gynecol 2005; 192 (05) 1458-1461
• 7 Bailit JL, Grobman WA, Rice MM. et al; Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD) Maternal-Fetal Medicine Units (MFMU) Network. Morbidly adherent placenta treatments and outcomes. Obstet Gynecol 2015; 125 (03) 683-689
• 8 Al-Zirqi I, Stray-Pedersen B, Forsén L, Daltveit AK, Vangen S. Uterine rupture: trends over 40 years. BJOG 2016; 123 (05) 780-787
• 9 Al-Zirqi I, Daltveit AK, Vangen S. Maternal outcome after complete uterine rupture. Acta Obstet Gynecol Scand 2019; 98 (08) 1024-1031
• 10 Al-Zirqi I, Daltveit AK, Vangen S. Infant outcome after complete uterine rupture. Am J Obstet Gynecol 2018; 219 (01) 109.e1-109.e8
• 11 Joseph KS, Young DC, Dodds L. et al. Changes in maternal characteristics and obstetric practice and recent increases in primary cesarean delivery. Obstet Gynecol 2003; 102 (04) 791-800
• 12 Habiba M, Kaminski M, Da Frè M. et al. Caesarean section on request: a comparison of obstetricians' attitudes in eight European countries. BJOG 2006; 113 (06) 647-656
• 13 Murthy K, Grobman WA, Lee TA, Holl JL. Association between rising professional liability insurance premiums and primary cesarean delivery rates. Obstet Gynecol 2007; 110 (06) 1264-1269
• 14 Hauth JC, Owen J, Davis RO. Transverse uterine incision closure: one versus two layers. Am J Obstet Gynecol 1992; 167 (4 Pt 1): 1108-1111
• 15 Harrigill KM, Miller HS, Haynes DE. The effect of intraabdominal irrigation at cesarean delivery on maternal morbidity: a randomized trial. Obstet Gynecol 2003; 101 (01) 80-85
• 16 Walfisch A, Beloosesky R, Shrim A, Hallak M. Adhesion prevention after cesarean delivery: evidence, and lack of it. Am J Obstet Gynecol 2014; 211 (05) 446-452
• 17 Jauniaux E, Jurkovic D. Placenta accreta: pathogenesis of a 20th century iatrogenic uterine disease. Placenta 2012; 33 (04) 244-251
• 18 Ho CH, Lan CW, Liao CY, Hung SC, Li HY, Sung YJ. Mesenchymal stem cells and their conditioned medium can enhance the repair of uterine defects in a rat model. J Chin Med Assoc 2018; 81 (03) 268-276
• 19 Kim K, Bou-Ghannam S, Thorp H, Grainger DW, Okano T. Human mesenchymal stem cell sheets in xeno-free media for possible allogenic applications. Sci Rep 2019; 9 (01) 14415
• 20 Nakao M, Kim K, Nagase K, Grainger DW, Kanazawa H, Okano T. Phenotypic traits of mesenchymal stem cell sheets fabricated by temperature-responsive cell culture plate: structural characteristics of MSC sheets. Stem Cell Res Ther 2019; 10 (01) 353
• 21 Parekkadan B, Milwid JM. Mesenchymal stem cells as therapeutics. Annu Rev Biomed Eng 2010; 12: 87-117
• 22 Ra JC, Shin IS, Kim SH. et al. Safety of intravenous infusion of human adipose tissue-derived mesenchymal stem cells in animals and humans. Stem Cells Dev 2011; 20 (08) 1297-1308
• 23 Kurtz A. Mesenchymal stem cell delivery routes and fate. Int J Stem Cells 2008; 1 (01) 1-7
• 24 Braid LR, Wood CA, Wiese DM, Ford BN. Intramuscular administration potentiates extended dwell time of mesenchymal stromal cells compared to other routes. Cytotherapy 2018; 20 (02) 232-244
• 25 Devine SM, Cobbs C, Jennings M, Bartholomew A, Hoffman R. Mesenchymal stem cells distribute to a wide range of tissues following systemic infusion into nonhuman primates. Blood 2003; 101 (08) 2999-3001
• 26 Reinecke H, Murry CE. Taking the death toll after cardiomyocyte grafting: a reminder of the importance of quantitative biology. J Mol Cell Cardiol 2002; 34 (03) 251-253
• 27 Terrovitis JV, Smith RR, Marbán E. Assessment and optimization of cell engraftment after transplantation into the heart. Circ Res 2010; 106 (03) 479-494
• 28 Elman JS, Murray RC, Wang F. et al. Pharmacokinetics of natural and engineered secreted factors delivered by mesenchymal stromal cells. PLoS One 2014; 9 (02) e89882
• 29 Shimizu T. Cell sheet-based tissue engineering for fabricating 3-dimensional heart tissues. Circ J 2014; 78 (11) 2594-2603
• 30 Kanai N, Yamato M, Ohki T, Yamamoto M, Okano T. Fabricated autologous epidermal cell sheets for the prevention of esophageal stricture after circumferential ESD in a porcine model. Gastrointest Endosc 2012; 76 (04) 873-881
• 31 Takahashi H, Okano T. Thermally-triggered fabrication of cell sheets for tissue engineering and regenerative medicine. Adv Drug Deliv Rev 2019; 138: 276-292
• 32 Matsuura K, Haraguchi Y, Shimizu T, Okano T. Cell sheet transplantation for heart tissue repair. J Control Release 2013; 169 (03) 336-340
• 33 Miyahara Y, Nagaya N, Kataoka M. et al. Monolayered mesenchymal stem cells repair scarred myocardium after myocardial infarction. Nat Med 2006; 12 (04) 459-465
• 34 Hida N, Nishiyama N, Miyoshi S. et al. Novel cardiac precursor-like cells from human menstrual blood-derived mesenchymal cells. Stem Cells 2008; 26 (07) 1695-1704
• 35 Kaibuchi N, Iwata T, Yamato M, Okano T, Ando T. Multipotent mesenchymal stromal cell sheet therapy for bisphosphonate-related osteonecrosis of the jaw in a rat model. Acta Biomater 2016; 42: 400-410
• 36 Kawamura M, Miyagawa S, Fukushima S. et al. Xenotransplantation of bone marrow-derived human mesenchymal stem cell sheets attenuates left ventricular remodeling in a porcine ischemic cardiomyopathy model. Tissue Eng Part A 2015; 21 (15-16): 2272-2280
• 37 Kong P, Christia P, Saxena A, Su Y, Frangogiannis NG. Lack of specificity of fibroblast-specific protein 1 in cardiac remodeling and fibrosis. Am J Physiol Heart Circ Physiol 2013; 305 (09) H1363-H1372
• 38 Österreicher CH, Penz-Österreicher M, Grivennikov SI. et al. Fibroblast-specific protein 1 identifies an inflammatory subpopulation of macrophages in the liver. Proc Natl Acad Sci U S A 2011; 108 (01) 308-313
• 39 Patel AN, Vargas V, Revello P, Bull DA. Mesenchymal stem cell population isolated from the subepithelial layer of umbilical cord tissue. Cell Transplant 2013; 22 (03) 513-519
• 40 Tuma J, Carrasco A, Castillo J. et al. RESCUE-HF trial: retrograde delivery of allogeneic umbilical cord lining subepithelial cells in patients with heart failure. Cell Transplant 2016; 25 (09) 1713-1721
• 41 Bartolucci J, Verdugo FJ, González PL. et al. Safety and efficacy of the intravenous infusion of umbilical cord mesenchymal stem cells in patients with heart failure: a phase 1/2 randomized controlled trial (RIMECARD trial [randomized clinical trial of intravenous infusion umbilical cord mesenchymal stem cells on cardiopathy]). Circ Res 2017; 121 (10) 1192-1204
• 42 La Rocca G, Anzalone R, Corrao S. et al. Isolation and characterization of Oct-4+/HLA-G+ mesenchymal stem cells from human umbilical cord matrix: differentiation potential and detection of new markers. Histochem Cell Biol 2009; 131 (02) 267-282
• 43 Ishitani A, Sageshima N, Hatake K. The involvement of HLA-E and -F in pregnancy. J Reprod Immunol 2006; 69 (02) 101-113
• 44 Matsuura K, Utoh R, Nagase K, Okano T. Cell sheet approach for tissue engineering and regenerative medicine. J Control Release 2014; 190: 228-239
• 45 Sekine H, Shimizu T, Dobashi I. et al. Cardiac cell sheet transplantation improves damaged heart function via superior cell survival in comparison with dissociated cell injection. Tissue Eng Part A 2011; 17 (23-24): 2973-2980
• 46 Narita T, Shintani Y, Ikebe C. et al. The use of scaffold-free cell sheet technique to refine mesenchymal stromal cell-based therapy for heart failure. Mol Ther 2013; 21 (04) 860-867
• 47 Fukushima T, Uchiyama S, Tanaka H, Kataoka H. Hepatocyte growth factor activator: a proteinase linking tissue injury with repair. Int J Mol Sci 2018; 19 (11) E3435
• 48 Wilgus TA. Vascular endothelial growth factor and cutaneous scarring. Adv Wound Care (New Rochelle) 2019; 8 (12) 671-678
• 49 Kim SR, Yi HJ, Lee YN. et al. Engineered mesenchymal stem-cell-sheets patches prevents postoperative pancreatic leakage in a rat model. Sci Rep 2018; 8 (01) 360
• 50 Kato Y, Iwata T, Washio K. et al. Creation and transplantation of an adipose-derived stem cell (ASC) sheet in a diabetic wound-healing model, J Vis Exp. 2017 54539. 126
• 51 Kim K, Thorp H, Bou-Ghannam S, Grainger DW, Okano T. Stable cell adhesion affects mesenchymal stem cell sheet fabrication: effects of fetal bovine serum and human platelet lysate. J Tissue Eng Regen Med 2020; 14 (05) 741-753
• 52 Lee JK, Jin HK, Endo S, Schuchman EH, Carter JE, Bae JS. Intracerebral transplantation of bone marrow-derived mesenchymal stem cells reduces amyloid-beta deposition and rescues memory deficits in Alzheimer's disease mice by modulation of immune responses. Stem Cells 2010; 28 (02) 329-343
• 53 Djouad F, Bouffi C, Ghannam S, Noël D, Jorgensen C. Mesenchymal stem cells: innovative therapeutic tools for rheumatic diseases. Nat Rev Rheumatol 2009; 5 (07) 392-399
• 54 Bowers D, McKenzie D, Dutta D, Wheeless CR, Cohen WR. Growth hormone treatment after cesarean delivery in rats increases the strength of the uterine scar. Am J Obstet Gynecol 2001; 185 (03) 614-617