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US-20260124250-A1 - METHODS FOR SELECTING CULTURED CORNEAL ENDOTHELIAL CELLS

US20260124250A1US 20260124250 A1US20260124250 A1US 20260124250A1US-20260124250-A1

Abstract

The present invention provides a method for determining the quality of cultured corneal endothelial cells (CECs) comprising measuring in or on a cultured CEC a level of expression of the genes Thrombospondin-2 (THBS2) and/or Vitelline Membrane Outer Layer 1 Homolog (VMO1), and determining that the quality of said cultured CEC is non-therapy-grade if said measured level of expression corresponds to a THBS2 + and/or VMO1 + expression profile.

Inventors

  • Vanessa Lydia Simone LaPointe
  • Pere Catala Quilis
  • Mor Mordechai Dickman

Assignees

  • UNIVERSITEIT MAASTRICHT
  • ACADEMISCH ZIEKENHUIS MAASTRICHT

Dates

Publication Date
20260507
Application Date
20240129
Priority Date
20230127

Claims (18)

  1. 1 . A method for determining the quality of cultured corneal endothelial cells (CECs) comprising measuring in or on a cultured primary CEC a level of expression of the genes Thrombospondin-2 (THBS2) and/or Vitelline Membrane Outer Layer 1 Homolog (VMO1), and determining that the quality of said cultured primary CEC is non-therapy-grade if said measured level of expression corresponds to a THBS2+ and/or VMO1+ expression profile.
  2. 2 . The method of claim 1 , wherein said level of expression is measured as the absence or presence of the protein expression product of said genes, and wherein it is determined that the quality of said cultured CEC is non-therapy-grade if said protein expression product is detected, preferably wherein absence or presence of the protein expression product is measured by using fluorescently labeled antibodies.
  3. 3 . The method of claim 1 , further comprising measuring in or on said cultured CEC a level of expression of the gene Cingulin-like 1 (CGNL1), and determining that the quality of said cultured CEC is non-therapy-grade if said measured level of expression corresponds to a VMO1+, THBS2+, CGNL1− expression profile.
  4. 4 . The method of claim 1 , further comprising measuring in or on said cultured CEC a level of expression of at least one, preferably all, of CD166, CD56, CD44 and CD10, and determining that the quality of said cultured CEC is non-therapy-grade if said measured level of expression corresponds to a CGNL1 − , VMO1+, THBS2+, CD166 − , CD56 − , CD44+, and/or CD10+ expression profile, or determining that the quality of said cultured CEC is therapy-grade if said measured level of expression corresponds to a CGNL1+, VMO1 − , THBS2 − , CD166+, CD56+, CD44 − , and CD10 − expression profile.
  5. 5 . The method of claim 1 , further comprising measuring in or on said cultured CEC a level of expression of at least one gene selected from Amyloid Beta Precursor Protein (APP), S100 Calcium Binding Protein A10 (S100A10), Thioredoxin Reductase 1 (TXNRD1), Tropomyosin 2 (TPM2), Potassium Voltage-Gated Channel Subfamily E Regulatory Subunit 4 (KCNE4), Platelet Derived Growth Factor Receptor Alpha (PDGFRA), and 4-Hydroxyphenylpyruvate Dioxygenase (HPPD), and determining that the quality of said cultured CEC is non-therapy-grade if said measured level of expression corresponds to a APP − , S100A10±, TXNRD1+, TPM2+, KCNE4+, PDGFRA±, and/or HPPD expression profile, or determining that the quality of said cultured CEC is therapy-grade if said measured level of expression corresponds to a CGNL1+, VMO1 − , THBS2 − , CD166±, CD56±, CD44 − , CD10 − APR′, S100A10 − , TXNRD1 − , TPM2 − , KCNE4 − , PDGFRA − , and/or HPPD − expression profile.
  6. 6 . The method of claim 1 , wherein said cultured CEC is of human origin.
  7. 7 . The method of claim 1 , wherein said CEC is an individual CEC, more preferably wherein the level of expression is determined in or on a single cell.
  8. 8 . The method of claim 1 , wherein said measurement is performed on a cultured CEC at confluency in passages 0, 1, or 2.
  9. 9 . The method of claim 1 , wherein said measurement is performed on a cultured CEC cultured in the presence of a ROCK kinase inhibitor.
  10. 10 . The method of claim 1 , wherein the level of expression is measured by immunofluorescence techniques, preferably using fluorescently labeled antibodies.
  11. 11 . A method of excluding or selecting a culture of CECs for medical use, the method comprising: a) providing an in vitro culture of CECs, preferably cultured primary CECs or stem-cell-derived CECs, more preferably human CECs, b) performing the method of claim 1 on at least a part of said culture, and c) excluding said culture of CECs as a culture of CECs unsuitable for medical use when more than 10-15% of the cell population in said culture of CECs is of non-therapy-grade quality or when less than 85-90% of the cell population in said culture of CECs is of therapy-grade quality, or selecting said culture of CECs as a culture of CECs for medical use when the cell population in said culture of CECs comprises CECs of which less than 10-15% is of non-therapy-grade quality or when more than 85-90% of the cell population in said culture of CECs is of therapy-grade quality.
  12. 12 . A method of producing a culture of CECs for medical use, comprising performing the method of claim 11 , wherein step c) comprises selecting said culture of CECs as a culture of CECs for medical use, and wherein said method further comprises the step of providing a culture of CECs by: harvesting the in vitro culture of CECs as culture of CECs for medical use when the incidence of therapy-grade quality CECs in said culture is at a desired level; or separating therapy-grade quality CECs from said in vitro culture of CECs and collecting the thus separated cells as a culture of CECs for medical use; or removing non-therapy-grade quality CECs from said in vitro culture of CECs and collecting the remainder of said culture as a culture of CECs for medical use.
  13. 13 . The method of claim 11 , wherein step a) comprises cultivating said CECs as a monolayer, optionally dissociating said monolayer, and providing CECs in said optionally dissociated monolayer with fresh tissue culture reagent to produce a subsequent passage, preferably wherein said cultivation occurs in the presence of a ROCK kinase inhibitor and/or a p53 inhibitor, and/or preferably wherein the CECs are cultivated in or on a substrate or scaffold.
  14. 14 . A culture of CECs, preferably in the form of a monolayer, wherein the cell population in said culture of CECs comprises CECs of which at least 85-90% is of therapy-grade quality, and of which at least 85-90% comprises a VMO1- and/or THBS2-expression profile, preferably wherein said culture is selected or produced by the method of claim 11 , and wherein said culture optionally comprises antibodies against a protein expression product of at least one gene selected from CGNL1, VMO1, THBS2, APP, S100A10, TXNRD1, TPM2, KCNE4, PDGFRA, and HPPD, preferably selected from CGNL1 and APP, most preferably CGNL1.
  15. 15 . A tissue engineered graft material comprising a culture of CECs according to claim 14 .
  16. 16 . A method for treating a patient suffering from corneal endothelial dysfunction comprising administering to said patient the culture of CECs of claim 14 , or a tissue engineered graft material comprising said culture of CECs.
  17. 17 . A computer-implemented method for determining the quality of cultured CECs comprising steps of: a) receiving data representing a level of expression of THBS2 and/or VMO1, preferably further including CGNL1, CD166, CD56, CD44 and CD10, said level of expression having been measured in or on at least one cultured CEC, and b) determining the quality of a cultured CEC based on comparing said data received under a) with reference data of the level of expression in a therapy-grade and/or non-therapy-grade reference CEC, wherein a CD166±, CD56±, CGNL1+, CD44 − , CD10 − , VMO1 − , and THBS2 − expression profile is indicative of the therapy-grade quality of said cultured CEC, and wherein a VMO1±, THBS2+, CGNL1 − , CD166 − , CD56 − , CD44±, and/or CD10±, expression profile is indicative of the non-therapy-grade quality of said cultured CEC wherein step b) is preferably performed on a computer using an algorithm wherein the data received in step a) are compared with reference data, preferably wherein said reference values are stored on an accessible memory, and preferably wherein said algorithm can receive the data of step a).
  18. 18 . A data processing system comprising a processor adapted to perform the steps of the computer-implemented method of claim 17 , or a computer program product comprising instructions which, when the program is executed by a computer, cause the computer to carry out the computer-implemented method of claim 17 , optionally comprising a machine learning data processing model.

Description

CROSS REFERENCE TO RELATED APPLICATIONS This application is a 371 National Stage application of International Application No. PCT/NL2024/050038, filed Jan. 29, 2024, which claims the benefit of European Patent Application 23153812.5, filed Jan. 27, 2023, each of which is incorporated herein by reference in its entirety. FIELD OF THE INVENTION The present invention involves markers for selecting cultured corneal endothelial cells (CECs) that resemble native CECs to the exclusion of lower grade cells, markers for assessing the quality of cultured CECs, and methods of using the same. BACKGROUND OF THE INVENTION The cornea is the transparent front part of the eye that covers the iris, pupil, and anterior chamber transmitting light into the eye. The inner part of this avascular tissue is covered by a monolayer of hexagonal corneal endothelial cells (CECs) that maintain corneal transparency and hydration by their pump activity and barrier function. Human CECs are arrested in a non-proliferative state and lack regenerative capacity. Consequently, damage to CECs due to surgery or trauma, (inherited) diseases or acquired conditions that affect the corneal endothelium directly, inflammation and other ocular diseases may result in corneal endothelium dysfunction, leading to impaired vision or blindness. Corneal transplantation using donor tissue is the current therapy for treating corneal endothelium dysfunction. However, only one donor cornea is available for every 70 patients in need, leaving 12.7 million people awaiting treatment worldwide. The cultivation of primary CECs isolated through enzymatic digestion from a corneal endothelium and their expansion in culture has broken the one-donor-one-recipient paradigm, and clinical trials have shown that such primary cultivated CECs can restore corneal transparency. Encouraged by the long-term success of this therapy, clinical trials are ongoing in Japan, Mexico and Singapore to assess the therapeutic potential of cultured CECs. Nonetheless, the cultivation of CECs has limitations that prevent its wider adoption. At present, primary CEC cultures are only successful when derived from younger donors, e.g. younger than 45 years of age, limiting the pool of donor corneas suitable for this technique. Furthermore, in vitro expansion of primary CECs can presently only yield cells to a few passages with subsequent passaging resulting in dedifferentiation into fibroblast-like cells with loss of CEC phenotypic markers, typically surface markers such as cluster-of-differentiation (CD) antigens. Culture heterogeneity and significant alteration in phenotype and functionality of CECs is often observed after the second passage of cultured primary CECs. The low proliferation and rapid loss of phenotype of cultured primary CECs remains to be solved. Hence, alternative cell sources for CEC primary culture have been investigated, including induced pluripotent stem cells (iPSCs). Such developments hold great promise, but there is still a lack of clear parameters to identify cultured CECs that are of therapy-grade from cells that are of lower grade and unsuitable for transplantation. Surface markers such as CD166, CD44, CD105, CD24, CD26, and CD133, are frequently used as markers for evaluating the quality of cultured CECs, wherein CD166 is used as a positive marker for CECs and remaining markers are negative makers to exclude the fibroblastic-like phenotype (Toda et al., 2017. Investig. Ophthalmol. Vis. Sci. 58, 2011-2020). There is, however, still a need to identify additional or other cell-specific markers that allow to assess the quality of cultured CECs and to select and enrich for native-like CECs that can be used in therapy. SUMMARY OF THE INVENTION The present inventors used single-cell RNA sequencing (scRNA-Seq) to profile 42,220 primary human CECs from six corneas of three donors at five time points over three passages in culture in order to deconstruct the heterogeneity and gain knowledge on the alterations arising from the primary culture of CECs. The inventors found that the culture diversified over time into heterogeneous subpopulations including cells less desirable for therapy that were entering a senescent or fibrotic state. The inventors identified markers that can be used alone or in combination to assess for native-like CECs and enrich for desired cell populations. Pseudo time analysis further uncovered the different trajectories arising during culture. Immunofluorescence analysis confirmed that several markers were exclusively expressed by good quality CECs and not expressed in cultures containing CECs with altered morphology, and that other markers were exclusively expressed by low quality CECs and not expressed in high quality CEC cultures. Together, the inventors' findings shed light on the various routes followed by CECs in culture, and provide novel markers to increase culture efficiency, to improve cultivation protocols, and to select a cell population for clinical