Corneal Repair and Immunosuppressive Effect After Intracameral Human Amniotic

Membrane Mesenchymal Stem Cells (hAM-MSCs) Application

Alejandro Navas, MD, MSc, Fátima S. Magaña-Guerrero, BSc, Pamela Martínez-Aboytes, MD,

Rodrigo Bolanos, MD, Enrique O. Graue-Hernández, MD, MSc, Yonathan Garfias, MD, PhD

Institute of Ophthalmology “Conde de Valenciana”, Mexico City, Mexico

dr.alejandro.navas@gmail.com

Financial Disclosure- AN: Carl Zeiss Meditec, Consultant (A); Alcon

Laboratories, Consultant (B) and STAAR Surgical, Consultant (B).

- FSMG, PMA, RB, EOGH & YG: No financial interest or relationship to disclose.

- Grants and support: Authors would like to thank National Council of Science and Technology (CONACYT #160286), and Institute of Ophthalmology “Conde de Valenciana” Patronage.

Purpose• To evaluate the effect of human amniotic

membrane mesenchymal stem cells (hAM-MSCs) over components of the innate immune system and their possible use in the regenerative ocular therapy.

Methods• Isolated hAM-MSCs were cultured in

DMEM/F12 with 10% fetal bovine serum (FBS). hAM-MSCs were characterized using anti-CD105, CD73, CD44, CD29 and CD45 antibodies by flow cytometry.

Methods

• In vitro tri-linage differentiation in hepatocytes, chondrocytes and neurons was performed. The xenotrasplant of hAM-MSCs was intracamerally performed in a murine corneal burn model.

• The effect of hAM-MSCs on Neutrophil Extracellular Traps (NETs) were evaluated by flow microscopy. 

Figure 1: 3-millimeter diameter chemical burns were made. Different severity models were used: 1 NaOH, R-OH 99.6%

Figure 2: Simultaneous (immediately after injuries) intracameral injections of hAM-MSCs or BSS (sham) were performed.

Neutrophlis Extracellular Traps (NET’s)

Figure 3: NETs assays showing the capability of hA-MSCs to inhibit traps formation.

Results

• Cells with fibroblastic morphology and the ability to generate fibroblast-colony forming units were obtained. The phenotype of the cells was CD105+ CD73+ CD44+ CD29+ and CD45-, which suggests that these cells were mesenchymal cells.

• Their ability to differentiate into hepatocytes, chondrocytes and neurons was confirmed by the acquisition of albumin, collagen-II and nestin proteins, respectively.

Figure 5: Flow citometry characterization: CD 105, C D29, CD 73 Positive (+) and CD 34, CD45, CD 44 Negative (-).

Figure 6: hAM-MSc transdifferentiation assays. Collagen II (Insuline/chondrocytes) [A] and nestin (Tran retinoic acid/neurons [B].

A

B

Figure 4: Mesenchymal stem cells (hAM-MSCs) obtained from human amniotic membrane.

Results

• The xenotransplant of hAM-MSCs significantly improved the transparency of the murine damaged corneas and reduced the inflammatory infiltrate.

• On the other hand the interaction of peripheral blood polymorphonuclear cells (PBPMN) with the supernatant of hAM-MSCs decreased the NETs production.

Figure 7: NaOH = Sodium hydroxide 1N; BSS = Balanced Salt Solution; hA-MSCs = human Amniotic membrane-Mesenchymal Stem Cells. Burn murine model C57BL6 showing severe corneal vascularization, opacification and inflammation (A), an improvement in the intracameral hA-MSCs group was showed (B). Also severe cellular infiltration in the anterior chamber in the sham group (C) compared with mild reaction in the treated group (D). Fluoresence hA-MSCs marked are presented under fluoresence microscope (E,F). [DAPI nuclear counterstaining].

NaOH + BSS(Sham)

NaOH + intracameral hA-MSCs

Figure 8: R-OH = 99.6% alcohol; BSS = Balanced Salt Solution; hA-MSCs = human Amniotic membrane-Mesenchymal Stem Cells. A less severe murine burn model with absolute alcohol was performed. More vascularization and opacification were found in the sham group (A) compared with the hA-MSCs treated group (B) . H&E histological showed more inflammation and cellular infiltration in BSS (sham) gropu (C,E) compared with intracameral hA-MSCs treated group (D,F).

R-OH + BSS(Sham)

R-OH+ intracameral hA-MSCs

Conclusions• These data suggest that human Amniotic

membrane-Mesenchymal Stem Cells (hA-MSCs) could be applied in the ocular regenerative therapy as an immunosuppressor treatment in ocular and inflammatory diseases.

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