scholarly journals Planarians as an In Vivo Experimental Model for the Study of New Radioprotective Substances

Antioxidants ◽  
2021 ◽  
Vol 10 (11) ◽  
pp. 1763
Author(s):  
Artem M. Ermakov ◽  
Kristina A. Kamenskikh ◽  
Olga N. Ermakova ◽  
Artem S. Blagodatsky ◽  
Anton L. Popov ◽  
...  
Keyword(s):  
Rapid Development ◽  
Model System ◽  
Ionising Radiation ◽  
Genomic Technologies ◽  
Cell Pool ◽  
Dividing Cells ◽  
Dying Cells ◽  
First Time ◽  
Stem Cell Pool

Ionising radiation causes the death of the most actively dividing cells, thus leading to depletion of the stem cell pool. Planarians are invertebrate flatworms that are unique in that their stem cells, called neoblasts, constantly replace old, damaged, or dying cells. Amenability to efficient RNAi treatments, the rapid development of clear phenotypes, and sensitivity to ionising radiation, combined with new genomic technologies, make planarians an outstanding tool for the discovery of potential radioprotective agents. In this work, using the well-known antioxidant N-acetylcysteine, planarians are, for the first time, shown to be an excellent model system for the fast and effective screening of novel radioprotective and radio-sensitising substances. In addition, a panel of measurable parameters that can be used for the study of radioprotective effects on this model is suggested.

scholarly journals Hematopoietic Stem Cells Have an Intrinsic Expansion Limit

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 4749-4749
Author(s):  
Shanti Rojas-Sutterlin ◽  
André Haman ◽  
Trang Hoang
Keyword(s):  
Cell Cycle ◽  
Stem Cell ◽  
Chemotherapy Regimen ◽  
Gene Dosage ◽  
Hematopoietic Stem ◽  
Cell Functions ◽  
Cell Pool ◽  
Stem Cell Pool

Abstract Abstract 4749 Hematopoietic stem cell (HSC) transplantation is the first successful cellular therapy and remains the only treatment providing long-term cure in acute myeloblastic leukemia. At the apex of the hematopoietic system, quiescent HSCs are spared by chemotherapeutic treatments that target proliferating cells and therefore can regenerate the entire blood system of a patient after drug exposure. Nevertheless, the consequence of repeated chemotherapy regimen on HSC function remains to be clarified. We previously showed that Scl/Tal1 gene dosage regulates HSC quiescence and functions when transplanted at limiting dilutions (Lacombe et al., 2010). In the present study, we investigate how massive expansion in vivo influences stem cell functions. To address this question, we optimized a protocol based on 5-fluorouracil (5-FU), an antimetabolite that has been used to treat colon, rectum, and head and neck cancers. In addition, we used Scl+/− mice to address the role of Scl in controlling HSCs expansion post-5-FU. We show that within 7 days following 5-FU treatment, HSCs exit quiescence and enter the cell cycle. To deplete cycling HSCs, we injected a second dose of 5-FU and showed that the stem cell pool was disseminated. Nonetheless, the remaining HSCs proliferated extensively to re-establish the HSC pool, which was twice larger than that of untreated mice. At this point, most HSCs have exited the cell cycle and were back to quiescence. Despite a near normal stem cell pool size and a quiescent status, HSCs from these 5-FU treated mice could not compete against untreated cells to regenerate the host in transplantation assays. Furthermore, we show that this extensive proliferation in vivo severely impaired the clonal expansion of individual HSC as measured by the mean activity of stem cell (MAS). Our results demonstrate that HSCs lose their competitive potential after two 5-FU treatments, suggesting that HSCs have an intrinsic expansion limit beyond which their regenerative potential is impaired. In addition, Scl is haplodeficient for cell cycle entry and cell division but Scl gene dosage does not affect this expansion limit. Therefore, our data dissociate the control of HSC expansion under extensive proliferative stress from cell cycle control during steady state. We surmise that chemotherapy regimen based on repeated administration of 5-FU or other antimetabolites are likely to severely impair long-term stem cell functions. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Reactive Oxygen Species Registration in Planarian Regeneration

2015 ◽  
Vol 7 (6) ◽  
pp. 13 ◽  
Author(s):  
Kh. P. Tiras ◽  
S. V. Gudkov ◽  
V. I. Emelyanenko ◽  
K. B. Aslanidi
Keyword(s):  
Reactive Oxygen Species ◽  
Physiological State ◽  
Reactive Oxygen ◽  
Oxygen Species ◽  
Planarian Regeneration ◽  
The Difference ◽  
Dying Cells ◽  
First Time ◽  
Kinetics Of

<p class="1Body">Reactive oxygen species (ROS) are directly involved in cell proliferation, differentiation and apoptosis in a variety of organisms. We studied kinetics of own luminescence induced by changes of ROS in early stages of planarian regeneration. Kinetics of chemiluminescence were measured in intact planarians and the same individuals after decapitation within 15 hours. We analyzed the traumatic fluorescent signal obtained as the difference between kinetics of intact and decapitated planarians. It was found that regeneration is accompanied by changes in the content of ROS correlated with the energy-intensive process in regenerating planarians. Oxidative stress was caused by damage to cell membranes in the dissection of the planarian and it was accompanied by a drop in the intensity of luminescence with a time constant of about 3.6 hours. Phagocytosis of dying cells by neoblasts was accompanied by an increase of the luminescence intensity after 2 - 3 hours after decapitation. Neoblast mitosis was described by two maximums of luminescence over 5.1 hours and 8.3 hours after decapitation. For the first time we demonstrated the opportunity of registering the physiological state of pluripotent stem cells at the level of the organism <em>in vivo</em>.</p>

scholarly journals Embryo development and establishment of pregnancy after embryo transfer in pigs: coping with limitations in the availability of viable embryos

Reproduction ◽  
2002 ◽  
pp. 507-515 ◽  
Author(s):  
TJ King ◽  
J Zhu ◽  
HA Finlayson ◽  
W Bosma ◽  
L Harkness ◽  
...  
Keyword(s):  
Embryo Transfer ◽  
Zona Pellucida ◽  
Blastocyst Stage ◽  
Model System ◽  
Developmental Potential ◽  
Maintenance Strategies ◽  
First Time ◽  
Parthenogenetic Embryos

Embryo transfer and pregnancy maintenance strategies in pigs were evaluated with reference to situations in which limited numbers of viable embryos or micromanipulated embryos are available, such as pig cloning. Development of embryos with compromised zona pellucida was compared with development of embryos with intact zona pellucida. Micromanipulation had no effect on blastocyst production rates after development in vivo or in vitro, but development in vivo improved the number of embryos reaching the blastocyst stage. Transfer of embryos with compromised zona pellucida resulted in live piglets. Several hormone treatments to maintain pregnancy were tested in a model in which three embryos were transferred into unmated recipient gilts, compared with transfer of three embryos into mated recipients. None of the hormonal treatments resulted in pregnancy rates of more than 25% at term and no more than 9% of transferred embryos survived, in comparison with 50% of the mated recipients successfully carrying 25% of transferred embryos. Lastly, the developmental potential of parthenogenetic embryos was assessed and 62% of transferred embryos resulted in pregnancies, none of which continued beyond day 55 of gestation. After co-transfer of three fertilized embryos with 55-60 parthenogenetic embryos into each of six recipients, two live piglets were delivered. The results from the present study indicate that transfer of zona pellucida compromised embryos can yield litters of normal piglets. In addition, it was demonstrated in a model system involving the transfer of three fertilized embryos into mature gilts that hormonal pregnancy maintenance strategies support a low proportion of embryos to term. Lastly, the present study shows for the first time a comparably effective but novel alternative for pregnancy maintenance in the pig involving the co-transfer of parthenote embryos.

Hematopoietic Stem Cell Niche Interactions In The Embryo Can Modulate The Size Of The Stem Cell Pool Into Adulthood

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 585-585
Author(s):  
Owen J. Tamplin ◽  
Ellen M. Durand ◽  
Logan A. Carr ◽  
Pulin Li ◽  
Leonard I. Zon
Keyword(s):  
Stem Cell ◽  
Board Of Directors ◽  
Advisory Committees ◽  
Hematopoietic Stem ◽  
Cell Pool ◽  
Hsc Niche ◽  
Equity Ownership ◽  
Stem Cell Pool

Abstract Hematopoietic stem cells (HSC) reside in the bone marrow niche and sustain the production of blood throughout life. The entire pool of these rare and important cells is generated during a brief window of embryonic development. HSC are produced by the hemogenic endothelium of the dorsal aorta, migrate to and expand in the fetal liver, and then migrate again to seed the bone marrow. The zebrafish is a highly conserved and well-established model for HSC development. Similar to mammals, HSC emerge from the dorsal aorta, but then colonize a vascular plexus in the tail of the embryo—the caudal hematopoietic tissue (CHT). It is difficult to directly observe the interactions between an endogenous HSC and its niche, so we have developed the CHT as a model for HSC-niche interactions. To track HSC in vivo we have generated a transgenic reporter using the previously described mouse Runx1 +23 kb intronic enhancer. The purity of the stem cell pool marked by this reporter was determined. Using adult-to-adult limiting dilution transplantation with as few as one Runx1+23 positive cell, we have estimated the HSC purity to be approximately 1/35 (without immune matching), or similar to Kit+Sca1+Lin- (KSL) in mouse. This is the most pure stem cell population defined in the zebrafish system. Using embryo-to-embryo transplantation, a technique that is unique to zebrafish, we sorted Runx1+23 positive cells from one group of embryos and transplanted them to another by injection directly into circulation. Embryos are then grown to adulthood and marrow is tested for long-term engraftment between 3 and 5 months. This transplantation technique precedes formation of the thymus, thereby removing any chance of immune rejection. Highly stringent dilution of HSC in our embryo-to-embryo transplants has estimated a stem cell purity of one in two cells. Next, we applied our highly specific reporter to visualize HSC migration to the CHT niche. After arrival of the HSC, we have described 5 distinct steps during colonization: 1) adherence; 2) extravasation; 3) abluminal migration; 4) endothelial niche formation (“cuddling”); and 5) cell fate decisions. Live imaging analysis of HSC together with endothelial and stromal transgenic reporters has allowed us to quantify the relationship between different cell types within the CHT. For example, we observe preferential localization of HSC in close proximity to cxcl12a positive stromal cells. Lastly, we have sought to identify the molecular mechanisms involved in interactions between HSC and their niche. A chemical genetic screen identified the natural product lycorine as a small molecule that increases hematopoiesis in the CHT and promotes HSC-endothelial cell interactions. Combined chemical treatment and live imaging revealed that lycorine significantly increased the residence time of HSC in the niche. To test if treatment during the window of CHT colonization (2-3 days post fertilization) had long-term effects on HSC and the stem cell pool, the compound was washed off at 3 days and the Runx1+23 positive population was quantified by FACS. At 7 days post fertilization, after colonization of the marrow, there was a sustained and significant increase in Runx1+23 positive HSC. Strikingly, after 3 months, when treated embryos were raised to adulthood, we discovered that the increased HSC-endothelial cell interactions we observed in the CHT niche had in fact had an impact on the number of HSC in the adult. Our studies establish that the Runx1+23 transgenic is a highly specific reporter of HSC both in the embryo and adult, and that we can use this reporter for in vivo observation of an endogenous HSC niche. Furthermore, we show that the size of the adult stem cell pool can be altered by a transient signal during development. Disclosures: Tamplin: Boston Children's Hospital: Patents & Royalties. Zon:FATE Therapeutics, Inc: Consultancy, Equity Ownership, Founder Other, Membership on an entity’s Board of Directors or advisory committees, Patents & Royalties; Stemgent, Inc: Consultancy, Membership on an entity’s Board of Directors or advisory committees, Stocks, Stocks Other; Scholar Rock: Consultancy, Equity Ownership, Founder, Founder Other, Membership on an entity’s Board of Directors or advisory committees, Patents & Royalties.

scholarly journals Quiescence of hematopoietic stem cells and maintenance of the stem cell pool is not dependent on TGF-β signaling in vivo

2005 ◽  
Vol 33 (5) ◽  
pp. 592-596 ◽  
Author(s):  
Jonas Larsson ◽  
Ulrika Blank ◽  
Jenny Klintman ◽  
Mattias Magnusson ◽  
Stefan Karlsson
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Hematopoietic Stem Cells ◽  
Hematopoietic Stem ◽  
Cell Pool ◽  
Stem Cell Pool

Molecular Characterization of Stem Cell Populations and Specific Clone Tracking of Retrovirally-Marked Chemoprotected Stem Cells before and after Dose-Escalating Chemotherapy.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 291-291
Author(s):  
Brian C. Beard ◽  
Pau Mezquita ◽  
Laura J. Peterson ◽  
Tobias Neff ◽  
Ponni Anandakumar ◽  
...  
Keyword(s):  
Stem Cell ◽  
Peripheral Blood ◽  
Retroviral Vector ◽  
Post Transplant ◽  
In Vivo Selection ◽  
Cell Pool ◽  
Early Late ◽  
Dose Escalating ◽  
Stem Cell Pool

Abstract In vivo selection strategies of genetically modified cells carrying mutant forms of MGMT (i.e. P140K or G156A) have the potential to improve autologous and allogeneic stem cell gene therapy and transplantation. Previously, we have shown efficient in vivo selection and marrow protection in a clinically relevant canine model. Here we describe molecular analysis of stem cell pools before, during, and after aggressive chemotherapy with O6BG and BCNU or temozolomide. LAM-PCR was carried out to identify clones and track those clones over time. This study includes four dogs that received chemotherapy; two dogs received DLA-identical allogeneic transplants (G154/G069) and two dogs received autologous transplants (G197/G179). A control dog received an autologous transplant, but never received chemotherapy (G038) was included. Chemotherapy treated animals received CD34-enriched marrow cells transduced with an RD114-pseudotype retroviral vector encoding P140K and eGFP while the control dog retroviral vector encoded only eGFP. After stable engraftment four dogs were treated with either O6BG and BCNU or temozolomide. Initially, granulocyte marking levels ranged from 10–16% before the dogs received repeated dose-escalating regimens of chemotherapy. After the final round of chemotherapy granulocyte marking was &gt;98% and stabilized at 66–97% with stable increases in all cell lineages analyzed. For LAM-PCR analysis DNA was extracted from peripheral blood leukocytes before chemotherapy (71–95 days post-transplant designated PRE), after 4–6 treatments with chemotherapy (262–354 days post-transplant designated EARLY), and after the last treatment with chemotherapy (476–635 days post-transplant designated LATE). The DNA samples analyzed for G038 corresponded chronologically with that of the treatment animals with samples taken 95, 235, and 601 days post-transplant. Retroviral integration site analysis has shown that the four chemotherapy treated dogs and control dog are polyclonal at all times with no trend towards oligoclonal or monoclonal states. The stem cell pools of G038 are very similar with ~26% of clones identified in every pool after random sampling on bulk DNA. Additionally, overlapping sequences account for ~11% of PRE/EARLY clones, ~16% EARLY/LATE clones, and ~16% PRE/LATE clones. Unique sequences in each group account for about half of the clones analyzed. Conversely, in the dogs treated with chemotherapy, in over 40 clones analyzed no overlap between sequences has been identified. To further characterize the contribution of unique clones specific primers were designed to clones attained from integration sites collected in FACS-sorted populations from G197. When additional FACS-sorted and bulk peripheral blood samples from PRE/EARLY/LATE were probed the specific clone could be identified. This suggests that although the stem cell pool is polyclonal at all time points the clonal contribution of the pool is markedly different. This also suggests that the initial stem cell pool is composed of 100s to 1000s of clones. Retroviral copy number and telomere length of marked cells that have been selected by repeated treatments with chemotherapy is currently ongoing. Our studies suggest that even after aggressive in vivo selection of chemoprotected cells hematopoiesis has remained polyclonal and no side effects or malignancies were observed.

scholarly journals In vivo expansion of the circulating stem cell pool

Stem Cells ◽  
2009 ◽  
Vol 16 (S2) ◽  
pp. 131-138 ◽  
Author(s):  
Martin Körbling
Keyword(s):  
Stem Cell ◽  
Cell Pool ◽  
Stem Cell Pool

scholarly journals Proteinase 3 Is Expressed in Stem Cells and Regulates Bone Marrow Hematopoiesis

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 1159-1159
Author(s):  
Kutay Karatepe ◽  
Hongbo R. Luo
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Progenitor Cell ◽  
Caspase 3 ◽  
Bone Marrow Cells ◽  
Cell Compartments ◽  
Cell Pool ◽  
Stem Cell Pool

Abstract Previously published data from our lab identified proteinase 3 (PR3) as a key protein in regulating spontaneous death of neutrophils. Our lab showed that PR3 could cleave caspase 3 in a caspase 9-independent manner in ageing neutrophils (Loison et al. JCI. 2014). Meanwhile, the maintenance of stem cell pool in the bone marrow and the development of mature blood cells are tightly regulated processes. In an effort to understand the hematopoietic consequences of absence of PR3, we analyzed stem and progenitor cell compartments in wild-type (WT) and PR3 knock-out (PR3-/-) mice. Of note, PR3 is thought to be a neutrophil-specific serine protease. Surprisingly, we observed PR3 expression in sorted Lin- Sca-1+ c-Kit+ (LSK) cells by gene expression analysis at mRNA level as well as Western blotting at protein level. Conventional flow cytometry methods also confirmed this expression. While bone marrow cellularity was similar between WT and PR3-/- mice, conventional flow cytometry studies showed that bone marrow from PR3-/- mice contained a higher frequency of LSK and Lin- Sca-1- c-Kit+ (LK) cells. Colony forming cell assays also suggested that both bone marrow cells and splenocytes from PR3-/- mice contained a higher number of progenitor cells compared to their WT counterparts. Histological analysis of bone marrow smears revealed that bone marrow from PR3-/- mice had a higher number of immature myeloid cells and a reduced number of mature leukoyctes and lymphocytes. Next, we asked if the enhanced stem and progenitor cell compartments in PR3-/- mice were functional in vivo. Toward this aim, we performed competitive reconstitution experiments using total bone marrow cells as well as sublethal irradiation experiments. PR3-/- donor-derived cells gave rise to a higher number of cells in the recipient mice compared to WT donor-derived cells in competitive reconstitution experiments. Also, after WT and PR3-/- mice were irradiated with 4 Gy, peripheral blood cell counts recovered faster in PR3-/- mice. Similarly, when mice were irradiated with 6 Gy, PR3-/- mice had a higher survival rate. These data suggest that enhanced stem and progenitor cell compartments in PR3-/- mice are functional. To understand if the enhanced hematopoietic activity in PR3-/- mice is an intrinsic feature of stem cells, another set of competitive reconstitution experiments were performed using equal numbers of sorted LSK cells. Again, PR3-/- donor-derived cells were more dominant than WT donor-derived cells in the recipient mice. Then, we wanted to understand the underlying mechanism. The rate of proliferation and senescence in LSK cells were analyzed by BrdU incorporation studies and C12FDG staining, respectively. No difference in proliferation or senescence was observed between WT and PR3-/- mice. We also looked at the apoptosis rate of LSK cells in vivo and in vitro. Untreated LSK cells from WT and PR3-/- mice exhibited similar levels of viability both in vivo and in vitro. However, when bone marrow macrophages responsible for clearance of apoptotic cells were depleted using clodronate liposomes, PR3-/- LSK cells exhibit enhanced viability compared to WT LSK cells as evidenced by annexin V and 7-AAD staining. Using a fluorogenic substrate of caspase 3, we also observed that the enhanced viability seen in PR3-/- LSK cells after depletion of macrophages is due to a reduction in caspase 3 activation. Additionally, we asked whether LSK cells from PR3-/- mice represent features associated with stem cells from aged mice such as an increase in DNA damage accumulation and reduced polarity. Immunofluorescent microscopy studies suggested that long-term hematopoietic stem cells from PR3-/- mice exhibit higher levels of p-gH2AX foci, a marker of DNA damage, and a reduced polarized distribution of a-tubulin. Finally, we saw a reduced lifespan in PR3-/- mice. These data indicate that PR3 regulates bone marrow hematopoiesis by contributing to the maintenance of a healthy stem cell pool in the bone marrow. Disclosures No relevant conflicts of interest to declare.

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