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Stem Cell Research & Therapy Dec 2023Limbal 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...
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.
Topics: Humans; Stem Cells; Limbal Stem Cells; Limbus Corneae; Epithelium, Corneal; Spindle Apparatus; Endotoxins
PubMed: 38093301
DOI: 10.1186/s13287-023-03586-z -
BioRxiv : the Preprint Server For... Apr 2024Accurate positioning of the mitotic spindle within the rounded cell body is critical to physiological maintenance. Adherent mitotic cells encounter confinement from...
Accurate positioning of the mitotic spindle within the rounded cell body is critical to physiological maintenance. Adherent mitotic cells encounter confinement from neighboring cells or the extracellular matrix (ECM), which can cause rotation of mitotic spindles and, consequently, titling of the metaphase plate (MP). To understand the positioning and orientation of mitotic spindles under confinement by fibers (ECM-confinement), we use flexible ECM-mimicking nanofibers that allow natural rounding of the cell body while confining it to differing levels. Rounded mitotic bodies are anchored in place by actin retraction fibers (RFs) originating from adhesion clusters on the ECM-mimicking fibers. We discover the extent of ECM-confinement patterns RFs in 3D: triangular and band-like at low and high confinement, respectively. A stochastic Monte-Carlo simulation of the centrosome (CS), chromosome (CH), membrane interactions, and 3D arrangement of RFs on the mitotic body recovers MP tilting trends observed experimentally. Our mechanistic analysis reveals that the 3D shape of RFs is the primary driver of the MP rotation. Under high ECM-confinement, the fibers can mechanically pinch the cortex, causing the MP to have localized deformations at contact sites with fibers. Interestingly, high ECM-confinement leads to low and high MP tilts, which mechanistically depend upon the extent of cortical deformation, RF patterning, and MP position. We identify that cortical deformation and RFs work in tandem to limit MP tilt, while asymmetric positioning of MP leads to high tilts. Overall, we provide fundamental insights into how mitosis may proceed in fibrous ECM-confining microenvironments in vivo.
PubMed: 38659898
DOI: 10.1101/2024.04.12.589246 -
EMBO Reports May 2021In Caenorhabditis elegans zygote, astral microtubules generate forces essential to position the mitotic spindle, by pushing against and pulling from the cortex....
In Caenorhabditis elegans zygote, astral microtubules generate forces essential to position the mitotic spindle, by pushing against and pulling from the cortex. Measuring microtubule dynamics there, we revealed the presence of two populations, corresponding to pulling and pushing events. It offers a unique opportunity to study, under physiological conditions, the variations of both spindle-positioning forces along space and time. We propose a threefold control of pulling force, by polarity, spindle position and mitotic progression. We showed that the sole anteroposterior asymmetry in dynein on-rate, encoding pulling force imbalance, is sufficient to cause posterior spindle displacement. The positional regulation, reflecting the number of microtubule contacts in the posterior-most region, reinforces this imbalance only in late anaphase. Furthermore, we exhibited the first direct proof that dynein processivity increases along mitosis. It reflects the temporal control of pulling forces, which strengthens at anaphase onset following mitotic progression and independently from chromatid separation. In contrast, the pushing force remains constant and symmetric and contributes to maintaining the spindle at the cell centre during metaphase.
Topics: Animals; Caenorhabditis elegans; Caenorhabditis elegans Proteins; Microtubules; Spindle Apparatus; Zygote
PubMed: 33900015
DOI: 10.15252/embr.202050770 -
BioRxiv : the Preprint Server For... Jan 2023Mitosis is an essential process in which the duplicated genome is segregated equally into two daughter cells. CTCF has been reported to be present in mitosis but its...
Mitosis is an essential process in which the duplicated genome is segregated equally into two daughter cells. CTCF has been reported to be present in mitosis but its importance for mitotic fidelity remains to be determined. To evaluate the importance of CTCF in mitosis, we tracked mitotic behaviors in wild type and two different CTCF CRISPR-based genetic knockdowns. We find that knockdown of CTCF results in prolonged mitoses and failed anaphase segregation via time lapse imaging of SiR-DNA. CTCF knockdown did not alter cell cycling or the mitotic checkpoint, which was activated upon nocodazole treatment. Immunofluorescence imaging of the mitotic spindle in CTCF knockdowns revealed disorganization via tri/tetrapolar spindles and chromosomes behind the spindle pole. Imaging of interphase nuclei showed that nuclear size increased drastically, consistent with failure to divide the duplicated genome in anaphase. Population measurements of nuclear shape in CTCF knockdowns do not display decreased circularity or increased nuclear blebbing relative to wild type. However, failed mitoses do display abnormal nuclear morphologies relative to successful mitoses, suggesting population images do not capture individual behaviors. Thus, CTCF is important for both proper metaphase organization and anaphase segregation which impacts the size and shape of the interphase nucleus.
PubMed: 36712070
DOI: 10.1101/2023.01.09.523293 -
The Journal of Cell Biology Jan 2023Key for accurate chromosome partitioning to the offspring is the ability of mitotic spindle microtubules to respond to different molecular signals and remodel their...
Key for accurate chromosome partitioning to the offspring is the ability of mitotic spindle microtubules to respond to different molecular signals and remodel their dynamics accordingly. Spindle microtubules are conventionally divided into three classes: kinetochore, interpolar, and astral microtubules (kMTs, iMTs, and aMTs, respectively). Among all, aMT regulation remains elusive. Here, we show that aMT dynamics are tightly regulated. aMTs remain unstable up to metaphase and are stabilized at anaphase onset. This switch in aMT dynamics, important for proper spindle orientation, specifically requires the degradation of the mitotic cyclin Clb4 by the Anaphase Promoting Complex bound to its activator subunit Cdc20 (APC/CCdc20). These data highlight a unique role for mitotic cyclin Clb4 in controlling aMT regulating factors, of which Kip2 is a prime candidate, provide a framework to understand aMT regulation in vertebrates, and uncover mechanistic principles of how the APC/CCdc20 choreographs the timing of late mitotic events by sequentially impacting on the three classes of spindle microtubules.
Topics: Animals; Anaphase; Anaphase-Promoting Complex-Cyclosome; Cyclins; Microtubules; Spindle Apparatus; Cdc20 Proteins; Cyclin B
PubMed: 36269172
DOI: 10.1083/jcb.202203089 -
The Journal of Biological Chemistry Sep 2020The spindle apparatus segregates bi-oriented sister chromatids during mitosis but mono-oriented homologous chromosomes during meiosis I. It has remained unclear if...
The spindle apparatus segregates bi-oriented sister chromatids during mitosis but mono-oriented homologous chromosomes during meiosis I. It has remained unclear if similar molecular mechanisms operate to regulate spindle dynamics during mitosis and meiosis I. Here, we employed live-cell microscopy to compare the spindle dynamics of mitosis and meiosis I in fission yeast cells and demonstrated that the conserved kinesin-14 motor Klp2 plays a specific role in maintaining metaphase spindle length during meiosis I but not during mitosis. Moreover, the maintenance of metaphase spindle stability during meiosis I requires the synergism between Klp2 and the conserved microtubule cross-linker Ase1, as the absence of both proteins causes exacerbated defects in metaphase spindle stability. The synergism is not necessary for regulating mitotic spindle dynamics. Hence, our work reveals a new molecular mechanism underlying meiotic spindle dynamics and provides insights into understanding differential regulation of meiotic and mitotic events.
Topics: Metaphase; Microtubule-Associated Proteins; Schizosaccharomyces; Schizosaccharomyces pombe Proteins; Spindle Apparatus
PubMed: 32723864
DOI: 10.1074/jbc.RA120.012905 -
Frontiers in Genetics 2022Glioma is globally recognised as one of the most frequently occurring primary malignant brain tumours, making the identification of glioma biomarkers critically...
Glioma is globally recognised as one of the most frequently occurring primary malignant brain tumours, making the identification of glioma biomarkers critically significant. The protein KIF18A (Kinesin Family Member 18A) is a member of the kinesin superfamily of microtubule-associated molecular motors and has been shown to participate in cell cycle and mitotic metaphase and anaphase. This is the first investigation into the expression of KIF18A and its prognostic value, potential biological functions, and effects on the immune system and mitosis in glioma patients. Gene expression and clinicopathological analysis, enrichment analysis, and immune infiltration analysis were based on data obtained from The Cancer Genome Atlas (TCGA), with additional bioinformatics analyses performed. Statistical analysis was conducted in R software. Clinical samples were used to evaluate the expression of KIF18A via immunohistochemical staining. In addition, the expression level of KIF18A was validated on U87 cell line. Our results highlighted that KIF18A plays a key role as an independent prognostic factor in patients with glioma. KIF18A was highly expressed in glioma tissues, and KIF18A expression was associated with age, World Health Organization grade, isocitrate dehydrogenase (IDH) status, 1p/19q codeletion, primary therapy outcome, and overall survival (OS). Enrichment analysis revealed that KIF18A is closely correlated with the cell cycle and mitosis. Single sample gene set enrichment analysis (ssGSEA) analysis revealed that KIF18A expression was related to the immune microenvironment. The increased expression of KIF18A in glioma was verified in clinical samples and U87 cell line. The identification of KIF18A as a new biomarker for glioma could help elucidate how changes in the glioma cell and immune microenvironment promote glioma malignancy. With further analysis, KIF18A may serve as an independent prognostic indicator for human glioma.
PubMed: 35591854
DOI: 10.3389/fgene.2022.852049 -
EMBO Reports Jul 2021Progression through the cell cycle is driven by cyclin-dependent kinases that control gene expression, orchestration of mitotic spindle, and cell division. To identify...
Progression through the cell cycle is driven by cyclin-dependent kinases that control gene expression, orchestration of mitotic spindle, and cell division. To identify new regulators of the cell cycle, we performed transcriptomic analysis of human non-transformed cells expressing a fluorescent ubiquitination-based cell cycle indicator and identified 701 transcripts differentially expressed in G1 and G2 cells. Family with sequence similarity 110 member A (FAM110A) protein is highly expressed in G2 cells and localized at mitotic spindle and spindle poles during mitosis. Depletion of FAM110A impairs chromosomal alignment, delays metaphase-to-anaphase transition, and affects spindle positioning. Using mass spectrometry and immunoprecipitation, we identified casein kinase I (CK1) in complex with FAM110A during mitosis. CK1 phosphorylates the C-terminal domain of FAM110A in vitro, and inhibition of CK1 reduces phosphorylation of mitotic FAM110A. Wild-type FAM110A, but not the FAM110A-S252-S255A mutant deficient in CK1 phosphorylation, rescues the chromosomal alignment, duration of mitosis, and orientation of the mitotic spindle after depletion of endogenous FAM110A. We propose that CK1 regulates chromosomal alignment by phosphorylating FAM110A and promoting its interaction with mitotic spindle.
Topics: Anaphase; Cell Cycle Proteins; HeLa Cells; Humans; Mitosis; Phosphorylation; Spindle Apparatus
PubMed: 34080749
DOI: 10.15252/embr.202051847 -
Journal of Visualized Experiments : JoVE Nov 2022The fidelity of oocyte meiosis is critical for generating developmentally competent euploid eggs. In mammals, the oocyte undergoes a lengthy arrest at prophase I of the...
The fidelity of oocyte meiosis is critical for generating developmentally competent euploid eggs. In mammals, the oocyte undergoes a lengthy arrest at prophase I of the first meiotic division. After puberty and upon meiotic resumption, the nuclear membrane disassembles (nuclear envelope breakdown), and the spindle is assembled mainly at the oocyte center. Initial central spindle positioning is essential to protect against abnormal kinetochore-microtubule (MT) attachments and aneuploidy. The centrally positioned spindle migrates in a time-sensitive manner toward the cortex, and this is a necessary process to extrude a tiny polar body. In mitotic cells, spindle positioning relies on the interaction between centrosome-mediated astral MTs and the cell cortex. On the contrary, mouse oocytes lack classic centrosomes and, instead, contain numerous acentriolar MT organizing centers (MTOCs). At the metaphase I stage, mouse oocytes have two different sets of MTOCs: (1) MTOCs that are clustered and sorted to assemble spindle poles (polar MTOCs), and (2) metaphase cytoplasmic MTOCs (mcMTOCs) that remain in the cytoplasm and do not contribute directly to spindle formation but play a crucial role in regulating spindle positioning and timely spindle migration. Here, a multi-photon laser ablation method is described to selectively deplete endogenously labeled mcMTOCs in oocytes collected from Cep192-eGfp reporter mice. This method contributes to the understanding of the molecular mechanisms underlying spindle positioning and migration in mammalian oocytes.
Topics: Mice; Animals; Microtubule-Organizing Center; Spindle Apparatus; Sexual Maturation; Oocytes; Chromosome Segregation; Laser Therapy; Mammals
PubMed: 36440837
DOI: 10.3791/64439 -
Cell Death and Differentiation Feb 2021Entosis was proposed to promote aneuploidy and genome instability by cell-in-cell mediated engulfment in tumor cells. We reported here, in epithelial cells, that entosis...
Entosis was proposed to promote aneuploidy and genome instability by cell-in-cell mediated engulfment in tumor cells. We reported here, in epithelial cells, that entosis coupled with mitotic arrest functions to counteract genome instability by targeting aneuploid mitotic progenies for engulfment and elimination. We found that the formation of cell-in-cell structures associated with prolonged mitosis, which was sufficient to induce entosis. This process was controlled by the tumor suppressor p53 (wild-type) that upregulates Rnd3 expression in response to DNA damages associated with prolonged metaphase. Rnd3-compartmentalized RhoA activities accumulated during prolonged metaphase to drive cell-in-cell formation. Remarkably, this prolonged mitosis-induced entosis selectively targets non-diploid progenies for internalization, blockade of which increased aneuploidy. Thus, our work uncovered a heretofore unrecognized mechanism of mitotic surveillance for entosis, which eliminates newly born abnormal daughter cells in a p53-dependent way, implicating in the maintenance of genome integrity.
Topics: Aneuploidy; Breast Neoplasms; Entosis; Epithelial Cells; Gene Expression Regulation, Neoplastic; HEK293 Cells; Humans; MCF-7 Cells; Mitosis; Models, Genetic; Tumor Suppressor Protein p53; rhoA GTP-Binding Protein
PubMed: 33110215
DOI: 10.1038/s41418-020-00645-3