Expansion and characterization of human limbus-derived stromal/mesenchymal stem cells in xeno-free medium for therapeutic applications

Sahoo, Abhishek; Damala, Mukesh; Jaffet, Jilu; Prasad, Deeksha; Basu, Sayan; Singh, Vivek

doi:10.1186/s13287-023-03299-3

Cited by 4 publications

(2 citation statements)

References 85 publications

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“…The chondrogenic differentiation was identified by staining with 1% Alcian blue in 3% acetic acid solution (pH 2.5). 33 , 34 To quantify the extent of differentiation, the stains were eluted from the cells using the protocols previously described. 35 – 37 The intensity was measured using a UV-Vis spectrophotometer (SpectraMax M3; Molecular Devices, San Jose, CA, USA).…”

Section: Methodsmentioning

confidence: 99%

Human Lacrimal Gland Derived Mesenchymal Stem Cells – Isolation, Propagation, and Characterization

Jaffet

Mohanty

Veernala

et al. 2023

Invest. Ophthalmol. Vis. Sci.

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Purpose The existing treatment options for dry eye disease (DED) due to lacrimal gland (LG) dysfunction are mainly palliative. Mesenchymal stem cells (MSCs) based therapies and 3D-LG organoids have been explored as a curative option for LG regeneration in animal models. Human LG epithelial cultures are previously established and, here, we aim to isolate and characterize the spindle-shaped cells obtained from primary human LG cultures in order to unveil its MSC property. Methods Normal human lacrimal glands were obtained from individuals undergoing LG debulking surgery. The conditions for human LG-MSC culture were standardized to obtain pure population of LG-MSCs at passage 3. Population doubling time (PDT), expression of phenotypic markers, tri-lineage differentiation, colony forming potential, and gene expression analysis were carried out to assess the phenotypic and genotypic characteristics compared to bone marrow-MSCs (BM-MSCs). Results Our data show that these spindle-shaped cells exhibit similar phenotypic expression, colony-forming ability, and trilineage differentiation like BM-MSCs. Moreover, the gene expression also did not show any significant difference, except for increased IL1-β in LG-MSCs. The LG-MSCs do not express any lacrimal epithelial markers unlike LG tissue. Conclusions This study reveals the first-time evidence for the presence of MSC population within the human LGs, and these cells might play a role in maintaining healthy microenvironment within normal LG and repair in diseased LGs.

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Section: Methodsmentioning

confidence: 99%

Human Lacrimal Gland Derived Mesenchymal Stem Cells – Isolation, Propagation, and Characterization

Jaffet

Mohanty

Veernala

et al. 2023

Invest. Ophthalmol. Vis. Sci.

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“…where N is the number of live cells trypsinized in subcon uence, N 0 is the initial number of live cells seeded, and CT is the total number of culture days 22 .…”

Section: Cumulative Population Doubling and Doubling Timementioning

confidence: 99%

New characterization and safety evaluation of human limbal stem cells used in clinical application: fidelity of mitotic process and mitotic spindle morphologies

Zekušić,

Mihica,

Skoko

et al. 2023

Preprint

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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. Through a clinically applicable protocol,the collected data on LSCs at passage 1 were summarized: identity (cell size, morphology); potency (yield, viability, population doubling time, colony-forming efficiency were analyzed by trypan blue staining); expression of putative stem cell markers by 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.

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Crosstalk among miR‐29, α‐SMA, and TGFβ1/β3 in melatonin‐induced exosome (Mel‐prExo) treated human limbal mesenchymal stem cells (hLMSCs): An insight into scarless healing of the cornea

Altug,

Soykan,

Eyubova

et al. 2024

BioFactors

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Inflammatory mediators that infiltrate the corneal stroma after corneal infections, trauma or refractive surgery can trigger the transformation of corneal keratocytes into myofibroblasts, resulting in highly irregular collagen deposition and subsequently corneal scarring. Mesenchymal stem cells (MSCs) can be used as therapeutic agents to regenerate corneal and conjunctival tissue damage, regulate inflammation, and reduce the development of limbal stem cell failure. The use of MSC‐derived exosomes as a cell‐free therapeutic vector is a novel therapeutic approach. This study aimed to assess the effect of exosomes obtained from melatonin (Mel)‐treated human limbal mesenchymal stem cells (hLMSCs) on naïve hLMSCs and to determine their influence on the antifibrotic and pro‐regenerative pathways involved in corneal scarring. hLMSCs were treated with varying concentrations of Mel, followed by isolation and characterization of the procured exosomes (Mel‐prExos). These exosomes were added to the cell culture media of naïve hLMSCs to examine their antifibrotic and pro‐regenerative effects. The expression of miR‐155, miR‐29, TGFβ1, TGFβ3, PPARγ, and α‐SMA miRNAs and genes were compared between Mel‐treated hLMSCs and Mel‐prExo‐treated hLMSCs by using real‐time PCR. We found that at 1 μM Mel and in the presence of Mel‐prExos, TGFβ1 was expressed 0.001‐fold, while TGFβ3 was expressed 0.6‐fold. miR‐29 expression was increased 38‐fold in the control‐Exo group compared to that in the control group. Changes in TGFβ1/β3 and α‐SMA expression are associated with miR‐29 and miR‐155. This approach could prove beneficial for ocular surface tissue engineering applications.

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Expansion and characterization of human limbus-derived stromal/mesenchymal stem cells in xeno-free medium for therapeutic applications

Cited by 4 publications

References 85 publications

Human Lacrimal Gland Derived Mesenchymal Stem Cells – Isolation, Propagation, and Characterization

Human Lacrimal Gland Derived Mesenchymal Stem Cells – Isolation, Propagation, and Characterization

New characterization and safety evaluation of human limbal stem cells used in clinical application: fidelity of mitotic process and mitotic spindle morphologies

Crosstalk among miR‐29, α‐SMA, and TGFβ1/β3 in melatonin‐induced exosome (Mel‐prExo) treated human limbal mesenchymal stem cells (hLMSCs): An insight into scarless healing of the cornea

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