Sphere Formation Permits Oct4 Reprogramming of Ciliary Body Epithelial Cells into Induced Pluripotent Stem Cells

Ni, Aiguo; Wu, Ming Jing; Chavala, Sai H.

doi:10.1089/scd.2014.0064

Cited by 7 publications

(5 citation statements)

References 30 publications

(47 reference statements)

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“…These data suggest that the culture condition in spheroids has the effect of rejuvenating hCPCs, which acquire the potential of more cell cycles [ 56 ]. This observation is in line with what recently reported for cells from ciliary body, which, if in spheroids, could be reprogrammed in the presence of only Oct4, not requiring Klf4, which otherwise was necessary if cells were maintained as monolayers [ 57 ]. The fact that hCPCs are able to modulate positively and negatively telomerase depending on the culture conditions is noteworthy, since it indicates that the cells have not a tumoral behaviour but retain a stem cell profile.…”

Section: Discussionsupporting

confidence: 91%

Human Cardiac Progenitor Spheroids Exhibit Enhanced Engraftment Potential

et al. 2015

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A major obstacle to an effective myocardium stem cell therapy has always been the delivery and survival of implanted stem cells in the heart. Better engraftment can be achieved if cells are administered as cell aggregates, which maintain their extra-cellular matrix (ECM). We have generated spheroid aggregates in less than 24 h by seeding human cardiac progenitor cells (hCPCs) onto methylcellulose hydrogel-coated microwells. Cells within spheroids maintained the expression of stemness/mesenchymal and ECM markers, growth factors and their cognate receptors, cardiac commitment factors, and metalloproteases, as detected by immunofluorescence, q-RT-PCR and immunoarray, and expressed a higher, but regulated, telomerase activity. Compared to cells in monolayers, 3D spheroids secreted also bFGF and showed MMP2 activity. When spheroids were seeded on culture plates, the cells quickly migrated, displaying an increased wound healing ability with or without pharmacological modulation, and reached confluence at a higher rate than cells from conventional monolayers. When spheroids were injected in the heart wall of healthy mice, some cells migrated from the spheroids, engrafted, and remained detectable for at least 1 week after transplantation, while, when the same amount of cells was injected as suspension, no cells were detectable three days after injection. Cells from spheroids displayed the same engraftment capability when they were injected in cardiotoxin-injured myocardium. Our study shows that spherical in vivo ready-to-implant scaffold-less aggregates of hCPCs able to engraft also in the hostile environment of an injured myocardium can be produced with an economic, easy and fast protocol.

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Section: Discussionsupporting

confidence: 91%

Human Cardiac Progenitor Spheroids Exhibit Enhanced Engraftment Potential

et al. 2015

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“…Spheroid formation in ULAPs, as a classical method to form suspended 3D spheroids, is widely utilized for many cell types (Ni et al, 2014;Bu et al, 2020). Byun et al (2014) found that stemness transcription factor genes (NANOG and OCT4) were upregulated during suspended 3D spheroid culture in ULAPs.…”

Section: Discussionmentioning

confidence: 99%

Mechanotransduction Regulates Reprogramming Enhancement in Adherent 3D Keratocyte Cultures

Ding²,

Guo³

et al. 2021

Front. Bioeng. Biotechnol.

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Suspended spheroid culture using ultralow attachment plates (ULAPs) is reported to effect corneal fibroblast reprogramming. Polydimethylsiloxane (PDMS), with hydrophobic and soft substrate properties, facilitates adherent spheroid formation that promotes cellular physical reprogramming into stem-like cells without using transcription factors. However, it is still unknown whether the biophysical properties of PDMS have the same effect on adult human corneal keratocyte reprogramming. Here, PDMS and essential 8 (E8) medium were utilized to culture keratocyte spheroids and fibroblast spheroids, and the reprogramming results were compared. We provide insights into the probable mechanisms of the PDMS effect on spheroids. qPCR analysis showed that the expression of some stem cell marker genes (OCT4, NANOG, SOX2, KLF4, CMYC, ABCG2 and PAX6) was significantly greater in keratocyte spheroids than in fibroblast spheroids. The endogenous level of stemness transcription factors (OCT4, NANOG, SOX2, KLF4 and CMYC) was higher in keratocytes than in fibroblasts. Immunofluorescence staining revealed Klf4, Nanog, Sox2, ABCG2 and Pax6 were positively stained in adherent 3D spheroids but weakly or negatively stained in adherent 2D cells. Furthermore, OCT4, NANOG, SOX2, KLF4, HNK1, ABCG2 and PAX6 gene expression was significantly higher in adherent 3D spheroids than in adherent 2D cells. Meanwhile, SOX2, ABCG2 and PAX6 were more upregulated in adherent 3D spheroids than in suspended 3D spheroids. The RNA-seq analysis suggested that regulation of the actin cytoskeleton, TGFβ/BMP and HIF-1 signaling pathways induced changes in mechanotransduction, the mesenchymal-to-epithelial transition and hypoxia, which might be responsible for the effect of PDMS on facilitating reprogramming. In conclusion, compared to corneal fibroblasts, keratocytes were more susceptible to reprogramming due to higher levels of endogenous stemness transcription factors. Spheroid culture of keratocytes using PDMS had a positive impact on promoting the expression of some stem cell markers. PDMS, as a substrate to form spheroids, was better able to promote reprogramming than ULAPs. These results indicated that the physiological cells and culture conditions herein enhance reprogramming. Therefore, adherent spheroid culture of keratocytes using PDMS is a promising strategy to more safely promote reprogramming, suggesting its potential application for developing clinical implants in tissue engineering and regenerative medicine.

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“…Though experimental tumors have been created from ciliary body stem cells, in general, the term medulloepithelioma is reserved for embryonic neoplasms believed to arise from embryonal or fetal tissues, making ‘embryonal medulloepithelioma' redundant and ‘adult medulloepithelioma' contradictory [5,6,7]. At least 16 cases of medulloepithelioma have been reported in patients older than 20 years, with the oldest in a 79-year-old man [9,10].…”

Section: Discussionmentioning

confidence: 99%

Nonteratoid Medulloepithelioma Presenting in a 78-Year-Old Male

et al. 2016

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Background: Medulloepithelioma is a rare congenital neoplasm derived from precursors of the nonpigmented ciliary epithelium of the ciliary body. The average patient age at clinical presentation is 3.8 years. Case Presentation: We present the case of a 78-year-old male with progressive lens subluxation and ocular hypertension who was found to have a ciliary body mass. After enucleation for presumed ciliary body melanoma, histopathology showed a nonteratoid medulloepithelioma. Cytogenetic analysis revealed abnormalities in chromosomes 3 and 8. Conclusion: Medulloepithelioma is often initially misdiagnosed. Though congenital in nature, it can exhibit rapid growth, have chromosomal abnormalities, and must be considered in all age groups.

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Sphere Formation Permits Oct4 Reprogramming of Ciliary Body Epithelial Cells into Induced Pluripotent Stem Cells

Cited by 7 publications

References 30 publications

Human Cardiac Progenitor Spheroids Exhibit Enhanced Engraftment Potential

Human Cardiac Progenitor Spheroids Exhibit Enhanced Engraftment Potential

Mechanotransduction Regulates Reprogramming Enhancement in Adherent 3D Keratocyte Cultures

Nonteratoid Medulloepithelioma Presenting in a 78-Year-Old Male

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