Stem cells in hypometabolic state

INSPIRED BY DJAJA

Marija Vlaški & Zoran Ivanović

Etablissement Français du Sang Aquitaine-Limousin, Bordeaux/ U1035 INSERM-Université de Bordeaux

Everyone remembers the typical science-fiction movies when the astronauts wake up from hibernation after several hundred millions of miles and a couple of light-years upon arriving on destination.

Pure fantasy or feasible in reality?

Astounding physiological phenomenon of hibernation concerns the introduction of organisms into the state of a controlled metabolism in which the physiological functions are reduced to the minimum level, in parallel with the decrease of body temperature to the values approaching zero (1). Hibernating animals (polar squirrels, marmot, bears….) are able to develop a state of hibernation by developing hypoxia and hypercapnia, mainly by breathing control. This way, they can survive for several months, at temperatures approaching zero, and then return to the normal physiological normothermic state without damages (2).

In the first half of 20^th century, ingenious Serbian-French scientist Ivan Djaja and his followers faced the problem of hypothermia. The taboo was broken by their originality and brightness: they showed that under certain circonstances, it is possible to “force” a non-hibernating animal to hibernate (published with a delay: 3). The essential of their discovery is in the fact that the cooling of the animal should be done in a hypoxic and hypercapnic atmosphere (decreased O2 concentration and enhanced CO2 concentration with respect to ones in atmospheric air – 20%O2, 0.05% CO2). By this approach, the animals (rats) can survive cooling to around 0°C and then be rewarmed to the physiological body temperature without damages (4), while those cooled in atmospheric air O2 and CO2 concentrations died without exception.

Astounding, isn’t it?

Almost one century later, the lecture of Djaja and Andjus paper prompted us, two ex-students of a school that inherited Djaja’s tradition, to hypothesize that the stem cells, behaving as single celled primitive eukaryote, can be introduced in a hibernation state by applying the same principle: cooling in hypoxia and hypercapnia.

In fact, having in mind the properties of stem cells, this hypothesis seems to be plausible: the stem cells are “equipped” to survive and to divide without differentiation (self-renewal) in the state of metabolic dormancy (hypometabolism), with extremely low O2 demands (and availabilities) and even without “food” (state of ischemia) (5). These cells, upon physiological needs, “resuscitate”, becoming high proliferative capacity and differentiation cell-entities able to produce a huge number of mature cells belonging to the different cell lineages.

Applying analogy with Djaja’s “resuscitated” animals, the atmosphere of hypoxia and hypercapnia allowed to introduce the stem cells in the hypometabolic state, which, consequently resulted in their long-term survival at temperatures approaching zero enabling their conservation without a classical cryopreservation (freezing). In practical terms, our experiments showed that in atmosphere of hypoxia/hypercapnia, a cell population enriched in hematopoietic stem cells (CD34+ cells) survive without damages at+ 4°C three to four times longer than in standard protocols allowing their maintenance until 3 days maximum (6-8). Exposure to hypoxia/hypercapnia also greatly enhanced the survival of committed hematopoietic progenitors inside this CD34+ population (6-8), and maintained the full potential of hematopoietic stem cells i.e. their capacity to reconstitute the hematopoiesis in vivo (6,8).

However, working on this model we realized that for a full analogy with the natural animal hibernation as well as with Djaja’s and Andjus “artificial” hibernation, the hypoxia and hypercapnia should be paralleled with a gradual cooling, passing through a moderate hypothermic state before exposing the cells to severe hypothermia. This would have enabled hypoxia and hypercapnia to exert their biochemical effects allowing an efficient adaptation of the cell to severe hypothermia i.e. to induce controlled hypometabolic state and introducing it in hibernation state. The effects in question concern expressions of genes and proteins, which are essential to avoid hypothermic stress and damages. They still take on, although slowly, at moderately low temperatures (25 to 32°C). So, combined action of hypoxia, hypercapnia and moderate hypothermia should allow a biochemical preparedness leading to an adaptation to severe hypothermia. Applying this model, we demonstrated that the cells acquired resistance to hypothermic as well as to hyperthermic stress, last one provoked by their rewarming to 37°C during the shift from hypothermia (hibernation) to physiological state. Similar results were obtained with the cells of different ontogenic and evolutionary origin, suggesting that the mechanism of cell introducing in hibernation-like state is rather universal (9).

Our experiments are not yet completed: we are now looking for protein and genetic actors which are critical for this unique and unusual functional state of homeothermic organism cells conferring them the resistance to hypothermic stress, that we named “cell hibernation”.

References

1. Boutilier RG. 2001. Mechanisms of metabolic defense against hypoxia in hibernating frogs. Respir Physiol 128(3):365-377.
2. Storey KB, Storey JM. 2007. Tribute to P. L. Lutz: putting life on ‘pause’–molecular regulation of hypometabolism. J Exp Biol 210(Pt 10):1700-1714.
3. Giaja J, Andjus R. 1949. Sur l’emploie de l’anesthésie hypoxique en physiologie opératoire. CR Hebd Seances Acad Sci 229:1170-2.
4. Andjus RK, Smith AU. 1955. Reanimation of adult rats from body temperatures between 0 and + 2 degrees C. J Physiol 128(3):446-472.
5. Ivanovic Z , Vlaski-Lafarge M.2015. Elsevier Store: Anaerobiosis and Stemness, 1st Edition ISBN-9780128006115.
6. Jeanne M, Kovacevic-Filipovic M, Szyporta M, Vlaski M, Hermitte F, Lafarge X, Duchez P, Boiron JM, Praloran V, Ivanovic Z. Low-oxygen and high-carbon-dioxide atmosphere improves the conservation of hematopoietic progenitors in hypothermia. Transfusion. 2009 Aug;49(8):1738-46.
7. Ivanovic Z, Kovacevic-Filipovic M, Jeanne M, Ardilouze L, Bertot A, Szyporta M, Hermitte F, Lafarge X, Duchez P, Vlaski M, Milpied N, Pavlovic M, Praloran V, Boiron JM. CD34+ cells obtained from “good mobilizers” are more activated and exhibit lower ex vivo expansion efficiency than their counterparts from “poor mobilizers”. Transfusion. 2010 Jan;50(1):120-7.
8. Vlaski M, Negroni L, Kovacevic-Filipovic M, Guibert C, Brunet de la Grange P, Rossignol R, Chevaleyre J, Duchez P, Lafarge X, Praloran V, Schmitter JM, Ivanovic Z. 2014. Hypoxia/hypercapnia-induced adaptation maintains functional capacity of cord blood stem and progenitor cells at 4°C. J Cell Physiol. 229(12):2153-65.
9. Vlaski M, Ivanovic Z, Gerby S: Procédé de conservation des cellules et des tissus en hypothermie sévère (1 à8°C), ou l’hypercapnie seule avec un passage par l’hypothermie modéré (20 à35°C). Patent 1553659; 2015, France.