Marcelo Palacios
Chairman of the Scientific Committee of the International Society of Bioethics (SIBI)
Abstract
Twenty years have passed since Dolly the sheep was born by cloning (somatic cell nuclear transfer, SCNT) but the results of non-human mammalian cloning are very poor, and cause animal diseases and huge biological losses. So far the reprogramming of somatic cells shows very low rates of efficiency (~0.0006-1%) that have not improved in the last two decades of continuous research. I believe that the reprogramming errors are not the only cause of these low rates of cloning: the mammalian SCNT fails with a very high frequency mainly due to the damage that the technique itself inflicts in the egg and the somatic nucleus, and the very few successful cases occur only when the damage is not significant.
Introduction
True cloning performed by nuclear transfer from an adult and differentiated somatic cell to a previously enucleated egg (somatic cell nuclear transfer, SCNT), gives rise to a new cell, the nuclovulo (nucleus+ovum), distinct from the zygote because the sperm is not involved in its creation, while both can develop as embryos and give rise to offspring.
Prior to SCNT, the somatic cell (differentiated) must be reprogramed to a similar state of a pluripotent embryonic cell (undifferentiated) before the nucleus is extracted and transferred. Little is known yet about the reprogramming mechanisms. Their failures or incompletion (regulation and gene expression, epigenetic, etc.) are attributed to the inviability and losses detected in the laboratory as well as the pathologies observed during the animal´s pregnancy and offspring after birth or later. However, I believe that the technical manipulation itself, is the determinant factor of the failures.
“Despite immense promise, somatic cell reprogramming still faces a critical challenge. Specifically, every method described to date can be characterized by low efficiency rates ranging from ~0.0006-1%. The low rates of success, which have not improved after a decade* of intense research, limit development and application”. The rates, according to other authors, can be raised to 2-3 % and 5-10 % in cattle. (1)
* Note: It is more than a decade, because it has been 20 years since Dolly’s birth in 1996.
«The results are still not very encouraging in terms of its practical application. The cost is expensive and its efficiency is poor. With some exceptions, the few cloned individuals who manage to birth, show alterations in their development that surpasses the detected in individuals developed from the fertilized oocyte”. “In the case of reproductive cloning, the results obtained so far in animal models, showed a high rate of fetal mortality and alterations in the development of cloned individuals”.(2)
Despite the technological advances in SCNT during the last decade, and its scientific and medical importance, the molecular processes involved in nuclear reprogramming remain largely unknown and the overall efficiency of SCNT in mammals remains very low. The efficiency of cloning, defined as the proportion of transferred embryos that result in viable offspring, is approximately 2 to 3% for all species. However, in cattle, average cloning efficiency is higher than in other species, ranging from 5 to 20% [10 –15]. Among the factors thought to contribute to the greater success in cloning cattle are the relatively late embryonic genome activation specific for this species [16 –18] and the optimization of reproductive technologies, such as in vitro embryo production and embryo transfer, brought about by the cattle industry [19]. Additionally, the efficiency of nuclear transfer technology may be enhanced by better understanding the nature of reprogramming using the cow model, since approximately half of all SCNT’s worldwide are performed in this species [20].
Morbidity and mortality from SCNT
The objective reality is that hundreds of thousands of SCNT attempts were unsuccessful (without a global statistics for all of them). It is worth noting that sometimes, even with autopsy verification, the very high morbidity and mortality rates were observed:
– In the laboratory: large amount of nuclovulos had unviable development.
In a meeting in Washington (3 December 2001) the researcher Tanja Dominko presented the results of monkey cloning (Macacus rhesus) when she worked at the Regional Center of Research in Primates of Beaverton, Oregon (USA). The scientist had analyzed almost 300 embryos produced in three years, and although several seemed healthy, they all resulted inviable. When investigating the causes of this failure via cellular and molecular analysis of 2-cell zygotes and the successive cell divisions (blastomeres), all kinds of abnormalities were found. She described these abnormalities as a «museum of horrors»: multiple nuclei, transferred nucleus totally out of sync with the cytoplasm of the egg, abnormalities in the separation of the chromosomes, lack of chromosomes, loss of centrosomes, blastomeres with cancerous aspect, etc. Some came to the state of 32 cells, but they showed abnormalities in the cells, wich made them unfeasible. And she added: “A 1-2 percent success rate is not a success, it’s a biological accident” (3). What has changed between 1996 and 2017?
– Complicated pregnancies with malformations and tearing in the placenta, failures in development, serious aberrations, supernumerary organs, deformations of the umbilical cord, tumors, miscarriages, premature births, etc.
– After birth: offspring of great size (large offspring síndrome, LOS), or with hyperkalemia and immature lungs; or apparently normal offspring who died shortly after birth, with the spleen atrophied, premature aging (progeria), cancer, immune disorders and repair or acid-base metabolism, etc.
-Other pathologies observed in fetuses or offspring were: heart abnormalities, respiratory disorders and pulmonary hypertension, generalized edemas, stroke, pneumonia, infections, metabolic acidosis or hypothermia, serious deficiencies in the immune system, necrosis and fibrosis of the liver, kidney disorders, diabetes, nervous system problems, muscle atrophy, etc., without depleting the pathologic findings.
The failure of the SCNT for reproductive purposes in mammalians is evident, and it is globally recognized.
What are the causes of SCNT high rates of failure in non-human mammals?
Even with the advances in cellular research, and the renowned successes of some SCNTs, much emphasis has been placed on the failures being caused by reprogramming errors, forgetting that the mechanical aspects of the technique are crucial because they cause injury to the two structures involved: the somatic nucleus and, mainly, the egg to which it is transferred (4).
The egg and somatic cells are structures of great complexity, and when handled with SCNT in its various steps, its structural components suffer and their functions are altered. Note that SCNT is performed by aggressive media: (i), in order to remove the somatic nucleus, it is “torn” and disconnected from the organelles attached to it (the rugous endoplasmic reticulum), which alters the essential continuity of the pore channels in the membrane (nuclear pore complexes, NPC), the exchange pathway with the cytoplasm and its components; ii), in the egg, the membranes have to be drilled twice: one to take away the nucleus (10-12 % of its total mass), and a second one to introduce the somatic nucleus, which causes a collapse and an internal pressure. It is not surprising that piercing the egg causes damage to their organelles, to a greater or lesser extent: cytoskeleton, mitochondria, endoplasmic reticulum, Golgi apparatus, etc. It is very difficult, if not impossible, to accept that the manipulation of the egg and the nucleus does not cause morphological and functional disturbances within them, and that they are not linked to the malformations and diseases that occur due to SCNT.
Therefore SCNT, as invasive and plunder as it is, makes the created nuclovulo a injured cell, in which embryological development negative effects are to be expected, as it has been demonstrated with unviability, diseases and deaths. Remember:
-“ In the fruit fly, the elimination of the cytoplasm and its organelles of a inner segment or the translocation, produces changes in the location of limbs and organs, etc., with all kinds of serious malformations” (5). These experiences were also made with the zebrafish.
-“From a mechanical point of view the cytoskeleton causes the cell to behave as the architectural structures called Tensegrity Structures (tensional integrity)”. (6) Cytoskeleton integrity is essential for the proper cell functioning, that the SCNT disturbs.
-“The functions of the Golgi Apparatus are affected when its position and its structure in human cells change» and «also affects the formation of primary cilia, a sensor structure essential for the cellular functioning», because «this disturbance cause a multitude of diseases known as ciliopatie”. (7)
«A few weeks ago I decided to discontinue the use of the technique of nuclear transfer». The expert cloner claims that, despite some results, the technique is not efficient and wasted a lot of genetic material, so that «it is not profitable». «Given the low efficiency one ends up wondering how long the nuclear transfer will be considered a useful method». Ian Wilmut to the Daily Telegraph. 17 November 2007 (8)
Conclusions
The damage to the egg and the somatic nucleus structures and functioning caused by SCNT are important and play a fundamental role in the failures, pathologies and loss of genetic and biological materials and animal lives. I believe that the mammalian cloning fails with such a high frequency mainly due to the damage caused by the aggressive technique in the egg and the somatic nucleus, and that the few successes take place only when this damage is not significant.
Should we resign from reproductive cloning? It is within reason not to expect in the future an improvement on the very small percentage of successes unless non-traumatic procedures are applied when performing SCNT.
From an ethical point of view, the legitimate scientific enthusiasm cannot excuse the immense loss of biological and genetic material, the exorbitant volume of complications and the animal suffering caused by abnormal pregnancies and the pathological sequelae of the offspring.
References
(1) Eilertsen K: Dr. Eilertsen’s research interests and laboratory activities. Somatic Cell Nuclear Transfer. Somatic/Adult/Progenitor cell reprogramming. https://www.pbrc. edu/research-and-faculty. Pennington Biomedical Reseach Center. LSU.
-N. Rodríguez-Osorio, R.Urrego, J. B. Cibelli, K. Eilertsen, E. Memili: Reprogramming mammalian somatic cells Theriogenology 78(9) September 2012.
– Quince años después de Dolly, la ciencia revisa la clonación. El Mundo, A. López, 24 de Febrero 2012.
(2) Merchant Larios H: Clonación humana: implicaciones biológicas y éticas. Mensaje Bioquímico, Vol XXXII. Universidad Nacional Autónoma. DF, México (2008). (http://bq.unam. mx/mensaje bioquímico).
(3) -New Scientist (www.newscientist.com).
– In Cloning, Failure Far Exceeds Success G. Kolata dec. The New York Times 11, 12. 2001
-Dominko, T., Simerly, C., Navara, C., Payne, C., Capuano, S., Gosman G., Kowit-Yu, C., Takahashi, D., Chance, C., Compton, D., Hewitson, L., and Schatten, G.. Molecular Correlates of Primate Nuclear Transfer Failures. Science 300, 2003.
-Simerly, C., Navara, C., Hyun, S.H., Lee, B.C., Kang, S.K., Capuano, S., Gosman, G., Dominko, T., Chong, K.Y., Compton, D., Hwang, W.S., Schatten, G.. Embryogenesis and blastocyst development after somatic cell nuclear transfer in nonhuman primates: overcoming defects caused by meiotic spindle extraction. Dev. Biol. 276, 2004.
(4) Nüsslein-Volhard C., Wieschaus E.F.: Mutations affecting segment number and polarity in Drosophila. Nature, 1980.
-Anderson K.V., Jurgens G., Nüsslein-Volhard C.: Establishment of dorsal-ventral polarity in the Drosophila embryo: Genetic studies on the role of the Toll gene product. Cell, 1985.
-Nüsslein-Volhard C., Dahm R: Zebrafish: a practical approach. Oxford University Press, 2002.
(5) Donald E. Ingber: Cellular tensegrity: defining new rules of biological design that govern the cytoskeleton. Journal of Cell Science 104, 1993.
–Donald E. Ingber, Laura Dike, Linda Hansen, Seth Karp y otros: Cellular Tensegrity: Exploring How Mechanical Changes in the Cytoskeleton Regulate Cell Growth, Migration, and Tissue Pattern during Morphogenesis. International Review of Cytology, Volume 150, 1994.
(6) Ríos R Mª y otros: La ubicación del aparato de Golgi en la célula es vital para el organismo Química.es Consejo Superior de Investigaciones Científicas (CSIC) el 17.06.2011, con referente en L. Hurtado, C. Caballero, M. P. Gavilán, J. Cárdenas, M. Bornens, and R. M. Rios Disconnecting the Golgi ribbon from the centrosome prevents directional cell migration and ciliogenesis, The Journal of Cell Biology, 30 de mayo 2011.
(7) Ian Wilmut now refuses nuclear transfer, and announces that their studies will focus on adult stem cells. Daily Telegraph 17.11.207 J. de la Varga Bioetica webs and Forum Libertas 19.11.07.
(8) Palacios, M.: La célula herida. La oveja Dolly y el nuclóvulo. Edit. Círculo Rojo, diciembre 2016.
-Palacios, Marcelo: ¿Por qué fracasa la clonación en humanos? Revista del Colegio Oficial de Médicos de Asturias. Abril 2017