Macrostomum ligano

Stem Cell Propagation in M.ligano

Basic Background

Macrostomum lignanoa free-living, hermaphroditic flatworm. It is transparent and relatively small in size with adults reaching about 1.7mm. M. lignano is mostly found in parts of the intertidal sand meiofauna of the Adriatic Sea.  It is an unsegmented, soft-bodied bilaterian missing a body cavity. All of them lack a specialized circulatory or respiratory organs.

Introduction.

Adult stem cells (ASCs) are responsible for self-renewal and the production of differentiated. They also have a high risk of malignant transformation due to the proliferative and error prone nature. It is theorized that ACSs might have acquired specialized features in order to protect the genome which is described as a stem cell trait. A mechanism by which this takes place was proposed as the immortal strand hypothesis.Label Retention Studies

Here, stem cells segregate DNA strands non-randomly upon asymmetric self-renewal. Sister chromatids with original strands of DNA are selectively retained in one daughter cell, which is destined to be renewed stem cell. Newly synthesized strands that possibly contain mutations are passed on to tissue-committed cells. This was observed using pulse-chase studies with nucleotide tracers: tritiated thymidine, bromodeoxyuridine (BrdU), or chlorodeoxyuridine (CldU). Pulse-chase studies use labeled compounds to follow the dynamics of cellular processes and pathways.  Labeling the original immortal strand during development or regeneration results in “Label-Retaining Cells” (LRCs).

Another method to retaining stem cells is cellular quiescence which is when control of stem cell division preserves genome integrity and prevents stem cell exhaustion. The low levels of proliferation reduces chances of label-dilution allowing more ASCs to be identified as LRCs. On the other hand, high levels of proliferation cause the label to dilute gradually leading to the process of double labeling protocol can be administered to avoid dilution.

It has been shown that the culturing environments have the ability to alter patterning of cells to modify fate and proliferative potential. Thus, models have gathered much attention due to this fact. M. lignano is a simple, triploblastic metazoans that exhibit powerful stem cell system which is maintained through adult life. They lies at the root of exceptional developmental plasticity and regeneration capacity. Their stem cell population comprised of pluripotent stem cells, referred to to as neoblasts (mitotically active during childhood, unlike all differentiated cells in the organism). They are highly advantageous model for in vivo stem cell research: easy to culture, short embryonic and post-embryonic development, and limited number of cells which facilitates cell quantification. The neoblasts can be well characterized and present in large number. Immunohistochemical staining of S-phase neoblasts with the thymidine analog bromodeoxyuridine and mitotic neoblasts with an anti-phospho histone H3 mitosis marker (anti-phos H3), have revealed a bilateral distribution of these cells.

The experiment.

M. lignano were cultured to obtain animals of standardized age by breeding adults for 24 hours from which the eggs were collected. Thymidine analog was administered to 11 age groups. The first five covered embryonic growth while the next six covered post-embryonic development. They were kept in standard culture medium, in the presence of food, 2-6 months in absence of BrdU. Simultaneous visualization of S-phase an mitotic neoblasts using anti-BrdU and anti-phos-H3 was performed. Rhodamine-conjugated goat-anti-rabbit was used as a secondary antibody for mitosis marker. The morphology of single cells were studied in which neoblasts were identified as small, rounded cells with large nucleus and scanty cytoplasm.

Macrostomum lignano

The same procedure was carried out with CldU and IdU which was double labeled on the standarized age groups of embryos and hatchlings. The animals were chased for 6 months with food and then pulsed. They were then visualized in the same fashion with anti bodies for the specific tracers. This was also carried out using EdU-pulse labeling and mounted to visualize the cells. Phase-contrast microscopy was used to see the mounted animals. A free software was used to quantify the BrdU LRCs.

Results.

To investigate if LRCs were present in M. lignano, thymidine analog was incorporated into the DNA of the animals by pulsing them with BrdU. After the specimens were chased for 2-6 months, 10 of each pulse-group were sacrificed in order to visualize and examine the presence of LRCs. 2 months of chasing showed presence of cells retaining BrdU label in both embryonic and post-embryonic groups. LRCs distributed all over the body. High density of BrdU cells was observed in bilateral pattern being in accordance with neoblast distribution. Another two clusters of labeled cells found: one near the brain and near mouth and pharynx. From 2 to 6 months, number of LRCs significantly lower in all groups: meaning cells were not able to retain label indefinitely or labeled cells replaced by progeny of unlabeled neoblast during tissue homeostasis.

  • 2 Months after Chase: 31 LRCs per animal
  • 6 Months after Chase: 13 LRCs per animal

Another experiment was conducted to see whether neoblasts could be determined within LRC population. To do this, a polyclonal antibody against a homolog of conserved Vasa protein of the model organism was utilized. In M. lignano, Macvasa is also present in a small set of somatic stem cells which shows up as a ring of spots surrounding the nucleus in the somatic cells. This allows Macvasa to be used as a marker. LRC were seen by using EdU and thus enabled Macvasa proteins to also be labeled.

After being pulsed continuously with EdU and then chased for three months with food to allow EdU-positive and Macvasa positive cells to be seen shown in Figure 4. The LRCs are labeled green.

Fig 4

Macvasa positive LRCs were found on the lateral side of the organism which was said to have contained some somatic neoblasts. Unfortunately, a downfall of this study, was that double positive cells were never observed in the testes or ovaries.

The proliferative activity of label-retaining stem cells was further analyzed by using a double labeling method of CldU and IdU. This was applied durin the S-phase of stem cells after 6 months of chase time. Proliferating LRCs was confirmed in every pulse group due to this. This proved that each 24-hour period of pulsing gave neoblasts that were able to retain the label for six months since neoblast are only somatic cells that are able to actively divide in M. lignano. Since they are always actively diving, no specific time frame of proliferation could be determined. A quantitative study showed the number of CldU/IdU labeled cells was low indicating low proliferative activity for label-retaining stem cells.

Cyochalasin D was used to test the segregation pattern of DNA strands in vivo which was determined to show binucleated cells. Equivalent EdU-signal in both daughter nuclei was observed. No cells were observed to show an unequal amount of fluorescence distribution.

Conclusion.

The four different approaches mentioned above were used to determine the separation mode of DNA-stands during stem cell division. All four of these approaches provided no evidence of non-random segregation of DNA making them inconsistent with the immortal strand hypothesis.

Fig 5

Long term label-retention served as confirmation of the existence of relatively quiescent stem cells. The data showed that LRCs were able to establish in M. lignano at 11 distince time period of development. These time periods included embryonic development, post embryonic development and even adulthood. A weakness of the study was that label retention was caused by an artificial removal from the cell cycle which was caused by deleterious effect of trying to incorporate thymidine. The double labeling experiment disproved this later due to proliferation of neoblasts. BrdU had no major effect on the cells as an analog. The effect of the analog incorporation was minimal and that label-retention was not caused by cell cycle arrest. Stem cells in M. lignano were identified based on three things:

  • Morphology
  • expression of Macvasa
  • ability to incorporate thymidine analog

The presence of label-retaining neoblast in LRCs was proven by three ways:

  1. Labeled neoblast identified morphologically
  2. Macvasa proteins demonstrated in subset of LRCs
  3. Small number of LRCs observed to incorporate IdU in double labeling

Observations showed that immortal strands would synthsize continuously during embryonic and post-embryonic development. Creating the new “immortal” strand is incompatible with the immortal strand hypothesis previously stated. Unfortunately, the exact timing of the development of LRCs is unable to be determined due to its compatibility with quiescent stem cells.

Cytochalasin D was used to prevent cytokinesis to analyze the distribution of labeled DNA among daughter cells of LRCs. Random segregation of the DNA-strands was seen among all the cells due to equal distribution of the labeled DNA. The results demonstrated that cellular quiescence was at large since it seemed to be present at the earliest stages of development. They were shown to remain in this stage for a significant amount of time as well.

This information allows researchers to further look into how the organization of the stem cell system is determined. Also, the analog which were utilized can be altered to provide different data that may suggest results other than those determined here.

References.

Verdoodt F, Willems M, Ladurner P, et al. Stem Cells Propagate Their DNA by Random Segregation in the Flatworm Macrostomum lignano. Plos One [serial online]. n.d.;7(1)Available from: Science Citation Index, Ipswich, MA. Accessed April 3, 2013.

“Pulse Chase.” New England BioLabs. N.p.. Web. 30 Apr 2013. <https://www.neb.com/applications/cellular-analysis/pulse-chase>.

Cairns J (2006) Cancer and the immortal strand hypothesis. Genetics 174:1069–1072.

Spradling A, Drummond-Barbosa D, Kai T (2001) Stem cells find their niche.Nature 414: 98–104.

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