New Characterization and Safety Evaluation of Human Limbal Stem Cells Used in Clinical Application: Fidelity of Mitotic Process and Mitotic Spindle Morphologies

Overview

Journal Stem Cell Res Ther

Publisher Biomed Central

Date 2023 Dec 14

PMID 38093301

Authors

Marija Zekusic

Marina Bujic Mihica

Marija Skoko

Kruno Vukusic

Patrik Risteski

Jelena Martincic

Iva M Tolic

Kreso Bendelja

Snjezana Ramic

Tamara Dolenec

Ivana Vrgoc Zimic

Dominik Puljic

Ivanka Petric Vickovic

Renata Ivekovic

Ivanka Batarilo

Ursula Prosenc Zmrzljak

Alan Hoffmeister

Tiha Vucemilo

Affiliations

Soon will be listed here.

Abstract

Background: Limbal stem cells (LSCs) are crucial for the regeneration of the corneal epithelium in patients with limbal stem cell deficiency (LSCD). Thus, LSCs during cultivation in vitro should be in highly homogeneous amounts, while potency and expression of stemness without tumorigenesis would be desirable. Therefore, further characterization and safety evaluation of engineered limbal grafts is required to provide safe and high-quality therapeutic applications.

Methods: After in vitro expansion, LSCs undergo laboratory characterization in a single-cell suspension, cell culture, and in limbal grafts before transplantation. Using a clinically applicable protocol, the data collected on LSCs at passage 1 were summarized, including: identity (cell size, morphology); potency (yield, viability, population doubling time, colony-forming efficiency); expression of putative stem cell markers through flow cytometry, immunofluorescence, and immunohistochemistry. Then, mitotic chromosome stability and normal mitotic outcomes were explored by using live-cell imaging. Finally, impurities, bacterial endotoxins and sterility were determined.

Results: Expression of the stemness marker p63 in single-cell suspension and in cell culture showed high values by different methods. Limbal grafts showed p63-positive cells (78.7 ± 9.4%), Ki67 proliferation (41.7 ± 15.9%), while CK3 was negative. Impurity with 3T3 feeder cells and endotoxins was minimized. We presented mitotic spindles with a length of 11.40 ± 0.54 m and a spindle width of 8.05 ± 0.55 m as new characterization in LSC culture. Additionally, live-cell imaging of LSCs (n = 873) was performed, and only a small fraction < 2.5% of aberrant interphase cells was observed; 2.12 ± 2.10% of mitotic spindles exhibited a multipolar phenotype during metaphase, and 3.84 ± 3.77% of anaphase cells had a DNA signal present within the spindle midzone, indicating a chromosome bridge or lagging chromosome phenotype.

Conclusion: This manuscript provides, for the first time, detailed characterization of the parameters of fidelity of the mitotic process and mitotic spindle morphologies of LSCs used in a direct clinical application. Our data show that p63-positive CK3-negative LSCs grown in vitro for clinical purposes undergo mitotic processes with extremely high fidelity, suggesting high karyotype stability. This finding confirms LSCs as a high-quality and safe therapy for eye regeneration in humans.

Citing Articles

Super-Resolution Imaging of Mitotic Spindle Microtubules Using STED Microscopy.

Koprivec I, Stimac V, Tolic I Methods Mol Biol. 2024; 2872:3-19.

PMID: 39616565 DOI: 10.1007/978-1-0716-4224-5_1.

References

Romano A, Espana E, Yoo S, Budak M, Wolosin J, Tseng S . Different cell sizes in human limbal and central corneal basal epithelia measured by confocal microscopy and flow cytometry. Invest Ophthalmol Vis Sci. 2003; 44(12):5125-9. DOI: 10.1167/iovs.03-0628. View

de Paiva C, Chen Z, Corrales R, Pflugfelder S, Li D . ABCG2 transporter identifies a population of clonogenic human limbal epithelial cells. Stem Cells. 2004; 23(1):63-73. PMC: 2906389. DOI: 10.1634/stemcells.2004-0093. View

Baba K, Sasaki K, Morita M, Tanaka T, Teranishi Y, Ogasawara T . Cell jamming, stratification and p63 expression in cultivated human corneal epithelial cell sheets. Sci Rep. 2020; 10(1):9282. PMC: 7283219. DOI: 10.1038/s41598-020-64394-6. View

Tseng S . Concept and application of limbal stem cells. Eye (Lond). 1989; 3 ( Pt 2):141-57. DOI: 10.1038/eye.1989.22. View

Hynds R, Bonfanti P, Janes S . Regenerating human epithelia with cultured stem cells: feeder cells, organoids and beyond. EMBO Mol Med. 2017; 10(2):139-150. PMC: 5801505. DOI: 10.15252/emmm.201708213. View

Deng S, Borderie V, Chan C, Dana R, Figueiredo F, Gomes J . Global Consensus on Definition, Classification, Diagnosis, and Staging of Limbal Stem Cell Deficiency. Cornea. 2019; 38(3):364-375. PMC: 6363877. DOI: 10.1097/ICO.0000000000001820. View

Prosenc Zmrzljak U, Kosir R, Krivokapic Z, Radojkovic D, Nikolic A . Detection of Somatic Mutations with ddPCR from Liquid Biopsy of Colorectal Cancer Patients. Genes (Basel). 2021; 12(2). PMC: 7923234. DOI: 10.3390/genes12020289. View

Fonseca C, Malaby H, Sepaniac L, Martin W, Byers C, Czechanski A . Mitotic chromosome alignment ensures mitotic fidelity by promoting interchromosomal compaction during anaphase. J Cell Biol. 2019; 218(4):1148-1163. PMC: 6446859. DOI: 10.1083/jcb.201807228. View

Sangwan V, Vemuganti G, Iftekhar G, Bansal A, Rao G . Use of autologous cultured limbal and conjunctival epithelium in a patient with severe bilateral ocular surface disease induced by acid injury: a case report of unique application. Cornea. 2003; 22(5):478-81. DOI: 10.1097/00003226-200307000-00016. View

10.

Menzel-Severing J, Kruse F, Schlotzer-Schrehardt U . Stem cell-based therapy for corneal epithelial reconstruction: present and future. Can J Ophthalmol. 2013; 48(1):13-21. DOI: 10.1016/j.jcjo.2012.11.009. View

11.

Wilhelm T, Olziersky A, Harry D, de Sousa F, Vassal H, Eskat A . Mild replication stress causes chromosome mis-segregation via premature centriole disengagement. Nat Commun. 2019; 10(1):3585. PMC: 6687892. DOI: 10.1038/s41467-019-11584-0. View

12.

Cotsarelis G, Cheng S, Dong G, Sun T, Lavker R . Existence of slow-cycling limbal epithelial basal cells that can be preferentially stimulated to proliferate: implications on epithelial stem cells. Cell. 1989; 57(2):201-9. DOI: 10.1016/0092-8674(89)90958-6. View

13.

Grueterich M, Espana E, Tseng S . Connexin 43 expression and proliferation of human limbal epithelium on intact and denuded amniotic membrane. Invest Ophthalmol Vis Sci. 2002; 43(1):63-71. View

14.

Hussain M, Fantuzzo R, Mercorelli S, Cullen C . A direct droplet digital PCR method for quantification of residual DNA in protein drugs produced in yeast cells. J Pharm Biomed Anal. 2016; 123:128-31. DOI: 10.1016/j.jpba.2016.01.050. View

15.

Lukinavicius G, Reymond L, DEste E, Masharina A, Gottfert F, Ta H . Fluorogenic probes for live-cell imaging of the cytoskeleton. Nat Methods. 2014; 11(7):731-3. DOI: 10.1038/nmeth.2972. View

16.

Santaguida S, Amon A . Short- and long-term effects of chromosome mis-segregation and aneuploidy. Nat Rev Mol Cell Biol. 2015; 16(8):473-85. DOI: 10.1038/nrm4025. View

17.

McIntosh J, Hays T . A Brief History of Research on Mitotic Mechanisms. Biology (Basel). 2016; 5(4). PMC: 5192435. DOI: 10.3390/biology5040055. View

18.

Kawashima M, Kawakita T, Satake Y, Higa K, Shimazaki J . Phenotypic study after cultivated limbal epithelial transplantation for limbal stem cell deficiency. Arch Ophthalmol. 2007; 125(10):1337-44. DOI: 10.1001/archopht.125.10.1337. View

19.

Ganem N, Godinho S, Pellman D . A mechanism linking extra centrosomes to chromosomal instability. Nature. 2009; 460(7252):278-82. PMC: 2743290. DOI: 10.1038/nature08136. View

20.

Roy A, Krzykwa E, Lemieux R, Neron S . Increased efficiency of gamma-irradiated versus mitomycin C-treated feeder cells for the expansion of normal human cells in long-term cultures. J Hematother Stem Cell Res. 2002; 10(6):873-80. DOI: 10.1089/152581601317210962. View