Mesenchymal Stem Cells
Mesenchymal stem cells are multipotent cells that can be obtained from umbilical cord tissue, adipose tissue1, dental pulp or amniotic liquid. The principal source of mesenchymal stem cells is the human bone marrow, which constitutes 4% of the total body mass of an individual2. They are able to differentiate to a variety of mesenchymal tissues lineages such as cartilage, fat, bone, muscle, tendon, and stromal tissue2.
This capacity to nourish tissue regeneration have positioned MSCs as promising medical treatments, with some studies already giving results in their applications in inflammatory bowel disease3 and other immune disorders4, or in ischemic heart disease5.
The initial enthusiasm, though, has been partly shadowed by the lack of a standardised and well-detailed cell processing and culture conditions protocols, which led to repeatability and scaling problems6.
Besides, mesenchymal stem cells are sensitive to experiment induced stresses such as phototoxicity or bleaching, present in fluorescence microscopy, the current method of choice for stem cells imaging. These types of stresses lead to a limitation in the cultured cells imaging possibilities. Implementation of the use of the 3D Cell Explorer microscope would help avoid these perturbations and improve this fundamental research as the samples need no preparation, which allows for a fast, non-invasive and expertise-independent live observation of mesenchymal stem cells. In addition, the 3D Cell Explorer laser uses 100 times less energy than the least energetic laser in the current fluorescent imaging approaches, which makes long-term imaging (up to weeks) possible.
In the provided example of a spectacular cell division taking place in a living sample of human mesenchymal stem cells kindly provided by Promocell Gmbh cultured with low-serum cell growth medium7 and observed under the 3D Cell Explorer. The characterization of the different steps and structures of mitosis was possible. Further details and static images of the high-quality footage obtained are here described (Figure 1).
Figure 1. The different phases of mitosis in Human Mesenchymal Stem Cells
Cell division is crucial in order to maintain an organism (Figure 2). To ensure growth, wound healing and replacement of damaged cells, eukariotic cells undergo mitosis (Figure 3).
A cell spends most of the duration of its life cycle in a stage known as interphase, which consists on three steps that prepair the cell for its division.
During G1 phase, the cell grows and it is metabolically active. It duplicates all its organelles but the chromosomes, which will be duplicated in the following step.
While in S phase, the synthesis of the cell DNA takes place. In the nucleus, the chromosomes are duplicated, and so are the centrosomes, a microtubule-organizing structure that plays a role in chromosome separation.
Figure 2. Cell cycle: stages and key events
The cell continues growing throughout the G2 phase, the phase that precedes mitosis. At this point there is a checkpoint, a cascade of signaling events that put replication on hold until any error found in the chromosomes resulting from the S phase is repaired (Figure 3).
This growth is evident in the first seconds of the video, where we notice that one of the four cells appears considerably bigger than the neighbouring ones.
That specific cell is in the G2 phase, the increase in size is due to the increase in the number of genes and gene products.
During late G2 phase, both the nuclear membrane and the nucleoli are intact. As we see in Figure 3 the nucleoli appear as dense and bright structures inside the nucleus surrounded by the chromatin, which looks like warped threads. It is exactly this unique thread shape which, in 1887, brought the German anatomist Walter Flemming to name this process after the Greek word for thread: mitosis.8
Figure 3. Signature structures of the cell in G2 phase
Once the cell is ready for division, it enters the first step of mitosis: the prophase. At this stage, the nucleoli disappear, and the chromatin condenses into chromosomes, two connected chromatids joined at the centromere (Figure 4)910.
Chromosome formation from disorganised chromatin will ease the separation into different cells in further steps. In this example, we were able to capture this transition from euchromatin to heterochromatin… live!
Figure 4. Signature structures of the cell in prophase
During prometaphase, the nuclear membrane breaks down into small-sized membrane vesicles (Figure 5) as the mitotic phase requires a wider space.
This open mitosis also involves an invasion of microtubules that will attach to the kinetochores (a disc-shaped structure associated to the chromosome chromatids), which will have a motor function helping centrosomes pull the chromosomes11–13.
Mitotic spindles work in conjunction with kinetochores, centrosomes and astral microtubules. Astral microtubules radiate from the centrosome towards the cell edge. All these structures work as a coordinated machinery, and cell organelles like mitochondria are usually absent in the area they delimit14,15. In the footage, we can observe a conserved structural integrity of mitochondria during this phase.
Figure 5. Signature structures of the cell in prometaphase
During metaphase and due to mitotic spindle’s tension, chromosomes line up along the metaphase plate. This plate is not a structure itself, but a term for the plane where the chromosomes line up as a result of an equally strong tension applied on the two sister chromatids from each chromosome, which are captured by the microtubules from opposite spindle poles (Figure 6).
An increase in refractive index at the metaphase plate is observed as a result of the big amount of proteins contained in the microtubules of the mitotic spindle and the kinetochores.
While all the steps until now follow a strict order, from now and until the complete separation of the daughter cells, the steps can overlap. That is, cytokinesis can happen simultaneously with anaphase and/or telophase16.
Figure 6. Signature structures of the cell in metaphase
During anaphase, chromosomes break at the centromeres and sister chromatids migrate to opposite ends of the cell (Figure 7).
The non-kinetochore spindle fibres, which are the microtubules that are not attached to the chromosomes, provide the tension needed for chromatid migration and work together with motor proteins to help chromosomes move along microtubule tracks.
Figure 7. Signature structures of the cell in anaphase
During telophase (Figure 8), the nuclear membrane will be formed to provide protection to the DNA. As the cell needs new gene products, the DNA needs to be accessible. DNA replication requires decondensed chromatin. For that reason, the chromosomes de-compact into chromatin, and the nucleoli containing ribosomes reappear.
Figure 8. Signature structures of the cell in telophase
Telophase follows or simultaneous to cytokinesis, which is not itself a phase of mitosis, but is needed to obtain the two separated daughter cells with a complete set of chromosomes, ready to enter G1 phase (Figure 9).
During this process, a complex structure called the contractile ring separates the two fully functional daughter cells, ending the cycle17.
Figure 9. Signature structures of the cell in cytokinesis
- Bunnell, B. A., Flaat, M., Gagliardi, C., Patel, B. & Ripoll, C. Adipose-derived stem cells: Isolation, expansion and differentiation. Methods 45, 115–120 (2008).
- Pittenger, M. F. et al. Multilineage potential of adult human mesenchymal stem cells. Science 284, 143–7 (1999).
- Research Supports Promise of Cell Therapy for Bowel Disease | Wake Forest Baptist Medical Center. Available at: https://newsroom.wakehealth.edu/News-Releases/2013/02/Research-Supports-Promise-of-Cell-Therapy-for-Bowel-Disease. (Accessed: 1st May 2019)
- Wang, M., Yuan, Q. & Xie, L. Mesenchymal Stem Cell-Based Immunomodulation: Properties and Clinical Application. Stem Cells Int. 2018, 1–12 (2018).
- Ward, M. R., Abadeh, A. & Connelly, K. A. Concise Review: Rational Use of Mesenchymal Stem Cells in the Treatment of Ischemic Heart Disease. Stem Cells Transl. Med. 7, 543–550 (2018).
- Robinson, P. G. et al. Reporting of Mesenchymal Stem Cell Preparation Protocols and Composition: A Systematic Review of the Clinical Orthopaedic Literature. Am. J. Sports Med. 47, 991–1000 (2019).
- Mesenchymal Stem Cell Growth Medium 2 | PromoCell. Available at: https://www.promocell.com/product/mesenchymal-stem-cell-growth-medium-2/. (Accessed: 6th May 2019)
- Paweletz, N. Walther Flemming: pioneer of mitosis research. Nat. Rev. Mol. Cell Biol. 2, 72–75 (2001).
- Shishova, K. V, Zharskaya, C. O. C. О. & Zatsepina, C. O. C. V. The Fate of the Nucleolus during Mitosis: Comparative Analysis of Localization of Some Forms of Pre-rRNA by Fluorescent in Situ Hybridization in NIH/3T3 Mouse Fibroblasts. Acta Naturae 3, 100–6 (2011).
- Cooper, G. M. The Nucleus during Mitosis. (2000).
- Maiato, H., DeLuca, J., Salmon, E. D. & Earnshaw, W. C. The dynamic kinetochore-microtubule interface. J. Cell Sci. 117, 5461–77 (2004).
- Heald, R. Motor function in the mitotic spindle. Cell 102, 399–402 (2000).
- Mcintosh, J. R. & Pfarr, C. M. Mini-Review Mitotic Motors. The Journal of Cell Biology 115, (1991).
- Ouellet, J. & Barral, Y. Organelle segregation during mitosis: lessons from asymmetrically dividing cells. J. Cell Biol. 196, 305–13 (2012).
- ROBBINS, E. & GONATAS, N. K. THE ULTRASTRUCTURE OF A MAMMALIAN CELL DURING THE MITOTIC CYCLE. J. Cell Biol. 21, 429–463 (1964).
- Mishima, M., Pavicic, V., Grüneberg, U., Nigg, E. A. & Glotzer, M. Cell cycle regulation of central spindle assembly. Nature 430, 908–913 (2004).
- Schroeder, T. E. The contractile ring. Zeitschrift f�r Zellforsch. und Mikroskopische Anat. 109, 431–449 (1970).