Dianne N. Irving
Copyright September 23, 2013
Reproduced with Permission
Note:
These scientific references are
not
provided to claim that the
separated
totipotent cells of the early human embryo -- or any
in vitro
induced "embryo-like" stem cells -- literally
are
embryos. Rather it is to scientifically document that many (not all) of these early human embryonic cells are
totipotent
-- and therefore
capable
of being reverted back to new whole embryos (if
"regulation" is successful") -- as happens in both natural and artificial monozygotic (MZ) twinning. "Regulation" can also
heal
damaged embryos when cells have been lost or removed. Hopefully these references will be found helpful. (Explanation of "regulation" in later article.)1
). [Emphases have been used below only to aid those unfamiliar with the science.]
I. Introduction
The term
"totipotent"
has
two
meanings.
First, it refers to the
single-cell human organism/human being/human embryo
sexually reproduced by fertilization -- whether done
in vivo
or
in vitro. This new single-cell human being is thus capable of producing
all
the cells, tissues and organs of an adult human being if allowed to fully develop.
Second, it refers to individual
cells
of the early developing human embryo. To say that many of these early cells (blastomeres) are totipotent is to say that IF they are separated from the original embryo then they are CAPABLE of being reverted back to new embryos IF the natural biological process of "regulation" is successful. If that process is not successful, then those separated totipotent cells remain as cells or simply die. Not new, as the following scientific references document (and they are just the tip of the iceberg). By contrast, the term
"pluripotent"
implies that, under
normal
conditions, the cell is
not
capable of undergoing regulation and being reprogrammed back to a new zygote - even if it were to be separated from the whole organism of which it is a part.
As documented below, many of the cells (blastomeres) of the early developing human embryo
before
implantation are totipotent - not pluripotent. Many of the cells of the early developing human embryo
after
implantation are also totipotent - not pluripotent. Precisely because these cells are totipotent -- not pluripotent -- and could undergo regulation,
monozygotic (identical) "twinning"
(one of many kinds of cloning) can take place, resulting in new asexually reproduced individual living human organisms. This can take place naturally
in vivo, or artificially
in vitro
- e.g., as you will see, by mechanically teasing the blastomeres apart (called "blastomere separation") or splitting the blastocyst in half (called "blastocyst splitting) - as is done in IVF and in ART research laboratories and "infertility" clinics. Both procedures are also referred to as "embryo multiplication".
Because these
early human embryonic cells
are also
diploid
(contain "46" nuclear chromosomes), they can also be cloned by nuclear transfer.
Embryonic germ line cells are also totipotent2
- not pluripotent -- and therefore can be cloned by "twinning".
Because these cells are
diploid,3
they can also
be cloned by nuclear transfer.
II. Scientific References:
The following selected bibliography on "totipotency" represents only the tip of the iceberg. Only a few of the thousands of items that came up on both PubMed (NIH) and Google searches are included here. The final reference list would be prohibitively long. But even these few references should make it clear to the reader that many of the cells of the early developing human embryo are totipotent - not pluripotent, as often claimed. Since the real experts on this issue are human embryologists, I have listed direct quotations from human embryology textbooks first.
A. Human Embryology Textbooks:
[Note:
These human embryology textbooks are obviously addressing the state of the early human embryo that exists
in vivo
-- within the woman's body. E.g., monozygotic (MZ) twinning -- also called "blastomere separation", "blastocyst splitting", "embryo multiplication" -- is one of many kinds of cloning. It takes place
naturally
while the developing embryo is still in the woman's fallopian tube traveling toward her uterus (about 5-6 days post-fertilization), as well as during and after implantation in her uterus. MZ twinning also takes place
artificially
in vitro
-- outside the woman's body in IVF and ART research laboratories and "infertility" clinics (usually used when older women have few "eggs" left). [Emphases have been used below only to aid those unfamiliar with the science.]
-
Ronan O'Rahilly and Fabiola Muller,
Human Embryology & Teratology
(3rd ed.) (New York: Wiley-Liss, 2001): "Biopsy of an embryo can be performed by removing one cell from a 4-cell, or two cells from an 8-cell, embryo. This does not seem to decrease the developmental capacity of the remaining cells. ...
The embryo enters the uterine cavity after about half a week, when probably at least 8-12 cells are present and the endometrium is early in its secretory phase.
Each cell (blastomere) is considered to be still totipotent (capable, on isolation, of forming a complete embryo), and separation of these early cells is believed to account for
one-third of cases of monozygotic twinning."
(p. 37)
-
O'Rahilly and Muller (2001), p. 136-137: "Stem cells comprise a small subpopulation of multipotent or pluripotent, ultrastructurally unspecialized, slow-cycling cells that possess the ability of self-renewal and can produce cells that are destined to differentiate. (In contrast, primordial germ cells and those of a morula are totipotent: i.e., they can develop into any type of embryonic tissues and can even form an entirely new embryo). Stem cells have a long proliferative reserve and are greatly influenced by their microenvironment. The signals that lead a stem cell to follow a specific developmental pathway are being investigated. When they become unipotent, i.e., committed to one line of development, the cells are generally known as progenitors and then precursors of differentiated cells. Stem cells are characteristic of all self-renewing tissues, such as the blood and the epidermis. Those in adult bone marrow include hematopoietic cells and mesenchymal cells; the latter can differentiate into fat, cartilage, bone, muscle, and possibly other types (e.g., hempatic cells). Dormant neural stem cells are present in the adult mammalian brain (e.g., in the ependyma, Rao, 10999) and are capable of forming neurons and glia. In addition to embryonic stem cells from preimplantation blastocysts, stem cells can be obtained from an adult. These latter possess remarkable potentialities; e.g., brain cells can become blood cells, and cells from bone marrow can become hepatic cells.
Ethical concerns are intensified by the experimental finding in primates (in contrast to the mouse) that
embryonic stem cells are totipotent and can develop into a complete embryo with a primitive streak."
-
Bruce Carlson,
Human Embryology & Developmental Biology
(St. Louis, MO: Mosby, 1999) (2nd ed.): "Mammalian embryogenesis is considered to be
a highly regulative process. Regulation is the ability of an embryo or an organ primordium to produce a normal structure if parts have been removed or added. [[i.e., regulation can "heal" a damaged embryo.]] At the cellular level, it means that the fates of cells in a regulative system are not irretrievably fixed and that the cells can still respond to environmental cues. Because the assignment of blastomeres into different cell lineages is one of the principal features of mammalian development, identifying the environmental factors that are involved is important. (pp. 44-49); ... Of the experimental techniques used to demonstrate
regulative properties of early embryos, the simplest is to
separate the blastomeres of early cleavage-stage embryos and determine
whether each one can give rise to an entire embryo. This method has been used to demonstrate that
single blastomeres, from two- and sometimes four-cell embryos
can form normal embryos, [[i.e., regulation can also revert totipotent embryonic cells back to new human organisms.]] ... (p. 44); ... Another means of demonstrating the
regulative properties of early mammalian embryos
is to dissociate mouse embryos into separate blastomeres and then to combine the blastomeres of two or three embryos.
The combined blastomeres soon aggregate and reorganize to become a single large embryo, which then goes on to become a normal-appearing tetraparental or hexaparental mouse.
By various techniques of making chimeric embryos,
it is even possible to combine blastomeres to produce interspecies chimeras
(e.g., a sheep-goat). (p. 45); ...
Blastomere removal and addition experiments have convincingly demonstrated the regulative nature (i.e., the strong tendency for the system to be restored to wholeness) of early mammalian embryos.
Such knowledge is important in understanding the reason
exposure of early human embryos to unfavorable environmental influences typically results in either death or a normal embryo. (p. 46); ...
Some types of
twinning
represent a natural experiment that demonstrates the
highly regulative nature
of early human embryos, ... (p. 48); ...
Monozygotic twins
and some triplets, on the other hand, are the product of one fertilized egg. They
arise by the subdivision and splitting of a single embryo. Although monozygotic twins could ... arise by the splitting of a two-cell embryo, it is commonly accepted that most arise by the subdivision of the inner cell mass in a blastocyst. Because the majority of monozygotic twins are perfectly normal,
the early human embryo can obviously be subdivided and each component regulated to form a normal embryo." (p. 49)
-
William J. Larsen,
Essentials of Human Embryology
(New York: Churchill Livingstone, 1998): [Monozygotic twinning
in humans] "If the splitting occurred during cleavage
-- for example, if the two blastomeres produced by the first cleavage division become separated -- the
monozygotic twin blastomeres will implant separately, like dizygotic twin blastomeres, and will not share fetal membranes. Alternatively,
if the twins are formed by splitting of the inner cell mass within the blastocyst, they will occupy the same chorion but will be enclosed by separate amnions and will use separate placentae, each placenta developing around the connecting stalk of its respective embryo. Finally,
if the twins are formed by splitting of a bilaminar germ disc, they will occupy the same amnion." (p. 325)
-
Moore and Persaud,
The Developing Human: Clinically Oriented Embryology
[Philadelphia: Saunders, 2003), 7th ed.]: [T]wins that
originate from one zygote
are monozygotic(MZ) twins or identical twins. The fetal membranes and placentas vary according to the origin of the twins. In the case of MS twins, the type of placenta and membranes formed depends
on when the twinning process occurs. (p. 144). ... MZ twinning usually begins in the
blastocyst stage, around the end of the first week, and results from
division of the embryoblast into two embryonic primordia. Subsequently, two embryos, each in its own aminiotic sac, develop
within the same chorionic sac and share a common placenta. ...
[E]arly separation of embryonic blastomeres (e.g., during the two- to eight-cell stages) results in MZ twins
with two aminions, two chorions, and two placentas that may or may not be fused. (p. 147) ... About
35% of MZ twins result from early separation of the embryonic blastomeres; i.e., during the first 3 days of development. The other 65 % of MZ twins originate at the end of the first week of development.
Late division of early embryonic cells, such as
division of the embryonic disc during the second week, results in MZ twins that are in one amnionic sac and one chorionic sac. ... If the embryonic disc does not divide completely, or adjacent embryonic discs fuse, various types of conjoined MZ twins may form. (p. 148) ...
Monozygotic twins .. represent about one-third of all twins; they are derived from one zygote.
(pp. 151, 153)
B. Human Genetics textbooks:
-
Tom Strachan and Andrew P. Read,
Human Molecular Genetics 2
(New York: John Wiley & Sons, Inc, 1999): "Animal clones occur
naturally
... For example,
genetically identical twins
are clones who happened to have received exactly the same set of genetic instructions from two donor individuals, a mother and a father. A form of
animal cloning can also occur as a result of
artificial
manipulation to bring about a type of
asexual reproduction. The genetic manipulation in this case uses
nuclear transfer technology: a nucleus is removed from a donor cell then transplanted into an oocyte whose own nucleus has previously been removed.
The resulting 'renucleated' oocyte can give rise to an individual
who will carry the nuclear genome of only one donor individual, unlike genetically identical twins.
The individual providing the donor nucleus and the individual that develops from the 'renucleated' oocyte are usually described as "clones", but it should be noted that they share only the same nuclear DNA;
they do not share the same mitochondrial DNA.
... Wilmut et al (1997) reported successful cloning of an adult sheep. For the first time,
an adult nucleus had been reprogrammed to become
totipotent
once more,
just like the genetic material in the fertilized oocyte
from which the donor cell had ultimately developed. ... Successful cloning of adult animals has forced us to accept that genome modifications once considered irreversible can be reversed and that
the genomes of adult cells can be reprogrammed by factors in the oocyte
to make them totipotent once again." (pp. 508-509)
-
Benjamin Lewin,
Genes VII
[Oxford: Oxford University Press and Cell Press, 2000), p. 605] [re
why germ line cells are initially totipotent]: A
change in methylation pattern
occurs during embryogenesis. All allelic
differences are lost
when
priordial germ cells
develop in the embryo; irrespective of sex,
the previous patterns of methylation are erased, and a typical gene is then
unmethylated. The methylation pattern of germ cells is therefore established by a two stage process: first
the previous pattern is erased by a genome-wide demethylation; then the
pattern specific for each sex is imposed.
C. IVF/ART textbooks:
-
Peter Brinsden (ed.),
A Textbook of In Vitro Fertilization and Assisted Reproduction, 2nd edition (New York: The Parthenon Publishing Group, 1999); Kay T. Elder, "Laboratory techniques: Oocyte collection and embryo culture", p. 197: "Surprisingly,
fragmented embryos, repaired or not, do implant and often come to term. This demonstrates the
highly robust nature
of the human embryo, as
it can apparently lose over half of its cellular mass and still recover."
-
Geoffrey Sher, Virginia Davis, and Jean Stoess,
In Vitro Fertilization: The A.R.T. of Making Babies
(copyright 1998 by authors; information by contacting Facts On File, Inc., 11 Penn Plaza, New York, NY 10001): "(2) the fertilized egg, which has not yet divided, is now known as a zygote; (3) the egg begins to divide and is now known as an embryo;
at this point each blastomere, or cell, within the embryo is capable of developing into an identical embryo." (p. 20)
D. Genuine Carnegie Stages Online:
-
STAGE 2:
http://www.medicalmuseum.mil/assets/documents/collections/hdac/stage02.pdf.
"Stage 2
comprises specimens
from 2 cells up to the appearance of the blastocystic (or segmentation) cavity. ... a
blastomere isolated
from the mammalian 2-cell organism is
capable of forming a complete embryo. Separation of the early blastomeres is believed to account for about one-third of all cases of monozygotic twinning in the human
(Corner, 1955). ...
Such twins should be dichorial and diamniotic
(fig. 5-2). The fact that nearly 60 percent of dichorial twins (whether monozygotic or dizygotic) have two unfused placentae 'indicates that the zona pellucida must have disappeared sufficiently long before implantation to allow the twins to become implanted in independent positions in the uterus' (Bulmer, 1970). Dizygotic twins, in contrast, are believed to arise from two oocytes, from a binucleate oocyte, or from a second polar body (Gedda, 1961). The successive cleavage divisions
do not occur synchronously, ... "
-
STAGE 3:
http://www.medicalmuseum.mil/assets/documents/collections/hdac/stage03.pdf.
"Stage 3
consists of the
free (that is, unattached) blastocyst, a term used as soon as a cavity (the blastocystic, or segmentation, cavity) can be recognized by light microscopy. ... The mammalian
blastocyst
differs from a blastula in that
its cells have already differentiated
into at least two types: trophoblastic and embryonic cells proper. Heuser and Streeter (1941) emphasized an important point by using stage 3 as an example:
The blastocyst form is not to be thought of solely in terms of the next succeeding stage in development. It is to be remembered that at all stages the embryo is a living organism, that is, it is a going concern with adequate mechanisms for its maintenance as of that time.
... It is no less true, however, that
changes occur in the growing organism and its environment
which provide critically for the future survival of the organism' (Reynolds, 1954). Indeed, such morphological and functional changes during development 'critically anticipate future morphological and functional requirements
for the survival and welfare of the organism'" (ibid.). ...
Duplication of the inner cell mass probably accounts for most instances of monozygotic twinning
(Corner, 1955; Bulmer, 1970). Such twins should be monochorial but diamniotic (fig. 5-2).
In Vitro, 'many blastocysts fail to hatch fully from their zona pellucida,' and 'two separate embryos could form if the inner cell mass was bisected during hatching' (Edwards, Mettler, and Walters, 1986)."
-
STAGE 5:
http://www.medicalmuseum.mil/assets/documents/collections/hdac/stage05.pdf.
"Stage 5
comprises embryos that are implanted to a varying degree but are previllous, i.e., that do not yet show definite chorionic villi. Such embryos are believed to be 7-12 days old. An amniotic cavity is found by stage 5.
If duplication of the embryo occurs after the differentiation of the amnion, the resulting monozygotic twins should be monochorial and monoamniotic (fig. 5-2). It has been estimated that the frequency of monoamniotic twins among monozygotic twins is about 4 percent (Bulmer, 1970). About once in every 400 monozygotic twin pregnancies, the duplication is incomplete and conjoined ('Siamese') twins (e.g., the second specimen of Shaw, 1932) result." ... Fig. 5-2. Diagram to illustrate the presumed mode of development of monozygotic twins in the human.
Based partly on Corner (1955). There exist ,
'three critical stages at which the division of the embryo to form monozygotic twins may occur'
(Bulmer, 1970). At
stage 2, before the differentiation of the trophoblast, separation of the blastomeres would result in twins with separate choria and amnia. At
stage 3 (and presumably at stage 4)
before differentiation of the amnion, duplication of the inner cell mass would result in twins with a common chorion but separate amnia. At
stage 5, duplication of the embryonic disc would result in twins with a common chorion and amnion. Deceptive fusion of the membranes may occur subsequently in certain instances but 'the placenta and membranes, if subjected to skilled examination, including microscopic study of the chorionamniotic walls when necessary, will generally yield a correct impression of the type of twinning' (Corner, 1955; see also Allen and Turner, 1971). Partial instead of complete embryonic separation would result in
conjoined twins
of the various types classified by Wilder (1904)."
E. Scientific Journals, Reports:
-
Scientists at the Danish Stem Cell Center, DanStem, at the University of Copenhagen have discovered that they can
make
pluripotent
embryonic stem cells regress to a stage of
totipotent
development
where these totipotent cells
are able to make placenta cells as well as the other fetal cells. By maintaining mouse embryonic stem cells under certain conditions, they found that
cells appear to regress and resemble
extremely early embryo cells
that can form any kind of cell including placenta and yolk sac cells. This significant discovery, published in the journal Cell Reports, has the potential to
shed new light on placenta related disorders that can lead to problematic pregnancies and miscarriages:
Sophie M. Morgani, Maurice A. Canham, Jennifer Nichols, Alexei A. Sharov, Rosa Portero Migueles, Minoru S.H. Ho, Joshua M. Brickman, "Totipotent Embryonic Stem Cells Arise in Ground-State Culture Conditions",
Cell Reports, Volume 3, Issue 6, 1945-1957, 06 June 2013; 10.1016/j.celrep.2013.04.034. at:
http://www.cell.com/cell-reports/fulltext/S2211-1247%2813%2900216-7. Embryonic stem cells (ESCs) are derived from mammalian embryos
during the transition from totipotency, when individual blastomeres can make all lineages, to pluripotency, when they are competent to make only embryonic lineages. ...
We observed heterogeneous expression of the extraembryonic endoderm marker
Hex
in 2i-cultured embryos, suggesting that 2i blocked development prior to epiblast commitment. Similarly, 2i ESC cultures were heterogeneous and
contained a
Hex-positive fraction primed to differentiate into trophoblast and extraembryonic endoderm. Single
Hex-positive ESCs coexpressed epiblast and extraembryonic genes and contributed to all lineages in chimeras. ... Thus,
2i and LIF support a totipotent state comparable to early embryonic cells that coexpress embryonic and extraembryonic determinants.
-
UNESCO, Report of the Eighteenth Session of the International Bioethics Committee (31 May - 2 June 2011), Baku, Azerbaijan;
Human Cloning and International Governance
(June 2, 2011), at:
http://www.unesco.org/new/fileadmin/MULTIMEDIA/HQ/SHS/pdf/Human-Cloning_IBC18_2011.pdf.
"The
terminology used in the bioethical debates is misleading
and does not adequately describe the technical procedures used (or potentially to be used) today. An in‐depth analysis aiming at re defining this terminology according to the new developments in human embryo research would be highly beneficial. ... The
existing international non‐binding texts
relevant to human cloning (i.e. the UNESCO Universal Declaration on the Human Genome and Human Rights of 1997 and the UN Declaration on Human Cloning of 2005)
are not sufficient to prevent human reproductive cloning. Possible ways of human reproductive cloning -
anno
2011: Embryo splitting; SCNT
- somatic cell nuclear transfer (FUSION of enucleated egg and somatic cell; DIRECT INJECTION of a nucleus into enucleated egg);
Tetraploid complementation
with EMBRYONIC STEM CELL;
iPS
‐ Induced Pluripotent Cells. Generation of EMBRYONIC STEM CELL - like cells (Tetraploid complementation; Direct derivation of sperm and egg cells). Many other methods of human reproductive cloning, in addition to SCNT, are already available." [[Three of the asexual reproductive techniques identified in this UNESCO Report are further explained with extensive scientific references in Irving,
"Any Human Cell - iPS, Direct Programmed, Embryonic, Fetal or Adult - Can Be Genetically Engineered to Asexually Reproduce New Human Embryos for Purposes of Reproduction ('Infertility')"
(November 2011), at:
http://www.lifeissues.net/writers/irv/irv_194cellasexuallyreproduce1.html]].
-
William Kearns, "Early embryos can correct genetic abnormalities
during development: findings have significant implications for fertility treatment and stem cell therapies", presented at the European Society of Human Reproduction and Embryology (July 3, 2011), at:
http://www.eshre.eu/membership/page.aspx/1354. See also, Amy Maxmen, "Embryos Right Genetic Wrongs? New evidence supports an old idea that
embryos with genetic abnormalities can somehow fix themselves early in development" (July 8, 2011), The Scientist, at:
http://the-scientist.com/2011/07/08/embryos-right-genetic-wrongs/.
-
Ferreira M.; Bos-Mikich, A.; Hoher, M.; Frantz, N.,
"Dichorionic twins and monochorionic triplets after the transfer of two blastocysts",
J. Assist. Reprod. Genet.
2010 Sep;27(9-10):545-8. Doi: 10.1007/s10815-010-9446-z. Epub 2010 Jul 28; PMID: 20665238; PMCID: PMC2965338.
Case report. A 24-year-old woman underwent ICSI and received
two blastocysts transferred. A
quintuplet gestation was established
. ... Three intrauterine gestational sacs were revealed at about 5th week. At the 7th week, five gestational sacs presenting heart beats were detected and a quintuplet pregnancy consisting of
two monozygotic (MZ) dichorionic twins and three MZ monochorionic triplets
was determined. At the 10th week, a single gestational sac with heart beats was detected. The prenatal course was uneventful.
A healthy baby was born at 36th week.
Few other reports have described the occurrence of
a quintuplet gestation after the transfer of two blastocysts generated by ICSI. Our case is unique in that the
two blastocysts underwent two different splitting processes, which occurred possibly at a similar time giving rise to MZ dichorionic twins and MZ monochorionic triplets.
-
Illmensee, K.; Levanduski, M.; Vidali, A.; Husami, N.; Goudas, VT,
"Human embryo twinning with applications in reproductive medicine", Fertil. Steril. 2010 Feb;93(2):423-7. doi: 10.1016/j.fertnstert.2008.12.098. Epub 2009 Feb 12; PMID: 19217091. To assess the
efficacy of human embryo twinning by blastomere biopsy at different early embryonic stages
(splitting efficiency) and to determine
the in vitro developmental capacity of twinned human embryos
(developmental efficiency). Randomized comparative study.
Private IVF centers. ... Embryos at the 2- to 5- and 6- to 8-cell stage were split into twin embryos.
Half the number of blastomeres from donor embryos were removed and inserted into recipient empty zonae pellucidae. After embryo splitting, donor and recipient embryos were cultured in vitro. The
number of developing embryos obtained after splitting could be increased
in comparison with the number of embryos available before splitting at the 6- to 8-cell stage but not at the 2- to 5-cell stage (splitting efficiency).
Splitting of 6- to 8-cell embryos yielded superior rates of twin embryos developing to blastocysts
(developmental efficiency). This is the first report on
twinned human embryos developing to blastocysts. This study exhibits the
potential for novel applications in human assisted reproduction.
-
Prukasananoda, K.; Rungsiwiwut, R.; Numchaisrika, P.; Ahnonkitpanich, V.; Virutamasen, P., "Development of human embryonic stem cell derivation",
J. Med. Assoc. Thai
2009, Apr;92(4):443-50; PMID: 19374291.
Abnormal and normal fertilization embryos were cultured
in vitro
until reaching blastocyst stage.
... The
feeder layers
used in the present study were fibroblasts, isolated from
either mouse or human. Mechanical splitting of ICM outgrowths or hES-like cells was performed for propagation of cells.
HES-like cells were spontaneously differentiated through suspension culture of embryoid body (EB). ... This is the first report in Thailand that
hES-like cells can be isolated from normal development human embryos at blastocysts-stage using mechanical isolation of ICM and culture with human adult skin fibroblast as feeder layers.
-
Van de Velde H, Cauffman G, Tournaye H, Devroey P, Liebaers I, "The
four blastomeres of a 4-cell stage human embryo are able to develop individually into blastocysts with inner cell mass and trophectoderm" (epub. May 24, 2008),
Hum Reprod.
2008 Aug;23(8):1742-7; PMID 18503052; at:
http://www.ncbi.nlm.nih.gov/pubmed/18503052?ordinalpos=107&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_DefaultReportPanel.Pubmed_RVDocSum
Early mammalian blastomeres are thought to be flexible and
totipotent
allowing the embryo to overcome perturbations in its organization during preimplantation development. In the past, experiments using single blastomeres from 2-, 4- and 8-cell stage mammalian embryos have provided evidence
that at least some of the isolated cells can develop into healthy fertile animals
and therefore are totipotent. We investigated whether isolated blastomeres of human 4-cell stage embryos could develop
in vitro
into blastocysts with trophectoderm (TE) and inner cell mass (ICM). ... The
majority of the blastomere-derived
embryos
followed the normal pattern of development with compaction on Day 4 and cavitation on Day 5 and developed into small blastocysts". They concluded that "the blastomeres of a 4-cell stage human embryo are flexible and able to develop into blastocysts with ICM and TE.
-
Bukovsky A, Svetlikova M, Caudle MR., "Oogenesis in cultures derived from adult human ovaries",
Reprod Biol Endocrinol. 2005 May 5;3(1):17 (http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=15871747&query_hl=7): "Development of numerous mature oocytes from adult ovarian stem cells in vitro offers new strategies for the egg preservation, IVF utilization, and treatment of female infertility. In addition,
other clinical applications aiming to utilize stem cells, and basic stem cell research as well, may employ
totipotent embryonic stem cells developing from fertilized oocytes."
-
DeRenzo C, Seydoux G, "A clean start: degradation of maternal proteins at the oocyte-to-embryo transition", Johns Hopkins School of Medicine,
Trends Cell Biol. 2004 Aug;14(8):420-6 [PMID: 15308208] (http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=15308208&query_hl=1: "In this article, we explore the hypothesis that the coordinated degradation of germline proteins is essential for
remodeling the oocyte into a
totipotent zygote
that is capable of somatic development."
-
Don P. Wolf, "Assisted reproductive technologies in rhesus macaques",
Reproductive Biology and Endocrinology, 2004, 2:37 [doi:10.1186/1477-7827-2-37] (http://www.rbej.com/content/2/1/37): "ICSI produced embryos were used in efforts to
create monozygotic twins by blastomere separation or blastocyst splitting.
While pregnancies were achieved following the transfer of
demi-embryos, only one was a twin and it was lost to spontaneous abortion.
ICSI produced embryos have also served as the source of blastocysts for the derivation of embryonic stem cells.
... This effort has also achieved the first twin pregnancies in rhesus monkeys, the first non-human primate infants produced by nuclear transfer of embryonic cells, the first rhesus monkey infant born following the transfer of an ICSI-produced blastocyst employing a non-surgical procedure,
the first monkey live birth resulting from the transfer of a
demi-embryo created by blastomere separation at the 2-cell stage or blastocyst bisection
and the first infants produced following laparoscopic embryo transfer. Recently we reported the outcomes of 87 pregnancies."
-
"Rhesus monkey is model for human embryonic stem cell research",
Reproduction and Development
(Sept. 17, 2004), Wisconsin National Primate Research Center, Univ. of Wisconsin-Madison (http://www.primate.wisc.edu/wprc/reproduction.html): "The utility of MHC-defined and
genetically identical animals
in a viral challenge model would have
tremendous impact on our ability to assess vaccine efficacy. This past year we have produced the first MHC-defined
rhesus monkeys
using assisted reproductive technologies. In addition, we have initiated studies to develop techniques for producing genetically identical MHC-defined monkeys
using blastomere separation, or embryo splitting. Using these techniques, we have produced genetically identical blastocysts
in vitro
at a relatively high rate. Continued improvements in assisted reproductive technologies in rhesus monkeys will enable us to develop a unique animal production program for the creation of MHC-defined and genetically identical monkeys
for use in immunological research and vaccine trials."
-
Christopher R. Cogle, MD; Steven M. Guthrie, BS; Ronald C. Sanders, MD; William L. Allen, JD, Edward W. Scott, Ph.D.; Bryon E. Petersen, Ph.D., "Stem Cell Research: An Overview of Stem Cell Research and Regulatory Issues",
Mayo Clin Proc.
2003;78:993-1003 (http://www.mayoclinicproceedings.org/article/S0025-6196(11)63146-7/fulltext): "As an extension of
research with embryonic stem cells, investigators have
also used these 'pluripotent' cells to produce identical offspring. Three approaches have been
used to
clone progeny. The first technique is blastomere separation. Splitting blastomeres at this
totipotent stage
leads to the development of genetically identical
offspring
but can produce only a limited brood due to the low cell number at the blastocyst stage."
-
[Cloning researcher] Geraedts JP, de Wert GM., "Cloning: applications in humans; Technical aspects",
Ned Tijdschr Tandheelkd. 2001 Apr;108(4):145-50]; [PMID: 11383357] (http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=11383357): "Cloning is possible by nucleus transplantation and by embryo splitting. Nucleus transplantation
does not result in a genetically completely identical individual because the mitochondrial DNA
originates from the ovum donor.
Embryo splitting
may be regarded as the artificial production of a monozygotic multiplet,"
-
C. Wood, "Embryo splitting: a role in infertility?" (July 17, 2001),
Reprod Fertil Dev, 13(1):91-3; PMID 11545169; at: http://www.ncbi.nlm.nih.gov/pubmed/11545169.
Embryo splitting may be used to increase the potential fertility of couples requiring IVF.
... The 30-40% greater chance of conception would reduce costs for the government, health authorities and patients, and reduce stress, time and complications for women having IVF treatment.
Embryo splitting
may also provide donor embryos for infertile couples that cannot conceive naturally or with IVF. The shortage of children for adoption and donor embryos may be overcome by the production of demi-embryos.
-
Sills ES, Tucker MJ, Palermo GD. (Georgia Reproductive Specialists LLC, Atlanta 30342, USA), "Assisted reproductive technologies and monozygous twins: implications for future study and clinical practice", 1:
Twin Res. 2000 Dec;3(4):217-23 (PMID: 11463142, at: http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=11463142&dopt=Abstract): "In vitro fertilization (IVF) relies on necessary (and, in some cases extended) embryo culture techniques potentially creating subtle ZP changes and subsequent
MZ [monozygotic] twinning. With growing experience in the assisted reproductive technologies and particularly IVF, some preliminary reports have noted an
increased frequency of MZ twins
after procedures that artificially breach the ZP (i.e., intracytoplasmic sperm injection [ICSI], or 'assisted hatching').
-
Geraedts JP, de Wert GM., "Cloning: applications in humans. I. Technical aspects" (May 13, 2000),
Ned Tijdschr Geneeskd, 144(20):921-6; PMID 10827846; at:
http://www.ncbi.nlm.nih.gov/pubmed/10827846. See also: Geraedts JP, de Wert GM., "Cloning: applications in humans. 1. Technical aspects" (April 2001),
Ned Tijdschr Tandheelkd, 108(4):145-50; PMID 11383357; at:
http://www.ncbi.nlm.nih.gov/pubmed/11383357. The successful cloning experiments in mammals such as the sheep and mouse prompted speculations on clinical application in humans.
Cloning is possible by nucleus transplantation and by embryo splitting.
Nucleus transplantation does not result in a genetically completely identical individual because the mitochondrial DNA originates from the ovum donor.
Embryo splitting may be regarded as the artificial production of a monozygotic multiplet.
Possible applications of cloning in humans belong
in the context of reproduction
(treatment of couples with subfertility, with genetic problems or with a 'replica motive'), transplantation of genetically identical tissue, and scientific research.
-
"Human Genome: The Book of Life",
Science, Volume 3 No.5 (September-November 2000) (http://www.iitk.ac.in/infocell/Archive/dirnov3/science.html): "Cloning: For Human Genome Project researchers, cloning refers to copying genes and other pieces of chromosomes to generate enough identical material for further study.
Two other types of cloning produce complete, genetically identical animals.
Blastomere separation creates identical twins (clones) which involves splitting a developing embryo soon after fertilization of the egg by a sperm to give rise to two or more embryos."
-
George J. Annas, Arthur Caplan & Sherman Elias, "Stem cell politics, ethics and medical progress",
Nature Medicine
5, 1339 - 1341 (1999) [doi:10.1038/70900] (http://www.nature.com/nm/journal/v5/n12/abs/nm1299_1339.html): "Tremendous controversy has surrounded efforts to undertake
research on
totipotent
human stem cells. To date public policy in the United States has attempted to skirt the ethical and social questions raised by this research. Annas et al. argue that
research using human embryos as
a source of totipotent stem cells
can secure broad public support if there is an open and public discussion about the ethical justification for undertaking such research and the assurance of adequate federal regulation and oversight."
-
DeMayo FJ, Rawlins RG, Dukelow WR, "Xenogenous and in vitro fertilization of frozen/thawed primate oocytes and blastomere separation of embryos",
Fertil Steril. 1985 Feb;43(2):295-300, (PMID: 3967788) (www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=3967788&dopt=Abstract): "Little research has been done on the
in vitro
and xenogenous fertilization of cryopreserved primate oocytes. This study reports the development of freezing and thawing methods for squirrel monkey oocytes with subsequent successful fertilization by these two methods.
Preliminary results on techniques for blastomere separation
using the hamster and squirrel monkey as models are also given. These studies have important implications relative to the long-term frozen storage of human oocytes, their subsequent thawing, in vitro fertilization and embryo transfer, and
the use of the blastomere separation procedure, in conjunction with in vitro fertilization, in the diagnosis of embryonic normality and possible congenital defects prior to implantation."
F. See also:
-
American Medical Association: "Human Cloning" (as of September 22, 2013), at:
http://www.ama-assn.org/ama/pub/physician-resources/medical-science/genetics-molecular-medicine/related-policy-topics/stem-cell-research/human-cloning.page. Scientists believe that the resultant cloning abnormalities are not traceable to the donor nuclei, but more likely explanations involve
failures in genomic reprogramming. ...
Methylation of DNA and other complex functions are now known to be essential to the correct functioning of each human cell, since they ultimately control gene expression. And thus successful cloning may be dependent upon the donated DNA being correctly altered to the state of an early embryo.
It is thought by some cloning experts that failure of the nuclear clones to produce viable offspring is due
to inappropriate reprogramming of cells, which leads to unregulated gene expression.
-
American Medical Association, Council on Ethics and Judicial Affairs (CEJA) Report 1-I-94: "Pre-Embryo Splitting" (http://www.ama-assn.org/resources/doc/ethics/ceja_1i94.pdf). [[CAUTION:
Note the AMA's use of the false scientific term "pre-embryo" in order to justify a range of procedures. Theologian Fr. Richard McCormick and frog embryologist Clifford Grobstein were at various times chairmen of the Ethics Committees of the AMA and several other related medical "professional societies". -- DNI]]
The first two techniques involve the procedure of
"splitting" or "twinning" embryos. "Blastomere separation" involves the division of a four-cell or eight-cell pre-embryo into the individual
totipotent
cells (blastomeres), or groups of such cells, which comprise it. ... A two-cell pre-embryo can be divided into two blastomeres, a four-cell pre-embryo into four blastomeres and an eight-cell pre-embryo into eight blastomeres. ... In effect, then,
the pre-embryo is "split" into its constituent cells which then may continue to develop. However there are important limitations to blastomere separation. It is often difficult to efficiently obtain human pre-embryos,
either by uterine flushing
or in vitro culture. ...
Once a blastocyst is obtained, its constituent cells rapidly lose their totipotency
so, given current scientific understanding, one pre-embryo can yield a maximum of four viable pre-embryos via blastomere separation. In addition,
many pre-embryos are destroyed in the process of separation. ... The
splitting of blastocysts
(multi-layered pre-embryos at the last stage before implantation) is referred to as "embryo splitting". In this technique,
a blastocyst is bisected into two multicellular groups of cells, each of which is nurtured to encourage further development. ...
JUSTIFICATIONS FOR PRE EMBRYO SPLITTING: Infertility
affects approximately 2.4 million married couples in the United States. A number of conditions may result in infertility: blocked or damaged fallopian tubes, ectopic pregnancies, ovulation problems, endometriosis, immunologic disorders, low sperm count, sexually transmitted diseases, and
advanced age. Although
adoption and
surrogacy
may be available to infertile couples, in vitro fertilization is often the only option that would allow both parents to contribute genetically to their child. If the technique is improved to the point of proven clinical efficacy and safety,
splitting of pre-embryos could serve as an important adjunct to in vitro fertilization by eliminating the need for the woman to undergo two kinds of medical risks. First, pre-embryo splitting can reduce the need to give drugs to the woman to stimulate ovulation.
Ordinarily, during the in vitro fertilization process, women are given drugs that stimulate ovulation in a way that produces multiple ova for retrieval; by transferring a few fertilized ova at one time to the woman's uterus, physicians can increase the chances that one of the pre-embryos will actually implant and develop into a child.
With pre-embryo splitting, since multiple pre-embryos can be obtained from one ovum, there might be less of a need to employ ovarian stimulant drugs with their various health risks such as possible increases in the rate of development of breast and ovarian cancer. ...
Second, pre-embryo splitting
reduces the need to subject women to the ovarian retrieval process. ... Pre-embryo splitting may
also facilitate the process of pre-implantation genetic diagnosis. In general, it is permissible for couples to screen pre-embryos for genetic disease.
If the pre-embryo is found to have such a disease, the couple can choose to discard or destroy the pre-embryo prior to implantation.
Preembryo splitting could be
used to create genetically identical pre-embryos destined for testing rather than transplant. This could increase the efficiency of pre-implantation diagnosis by either enabling all testing, with its attendant risks to the pre-embryo, to occur on a pre-embryo that was not destined for transplantation or by
doubling the amount of DNA available for genetic analysis. ... Apart from considerations about the potential benefits of pre-embryo splitting as a clinical procedure,
pre-embryo splitting may be an important research tool. Pre-embryo splitting could lead to the formation of
a genetically-identical pool of research subjects, an asset currently unavailable. This could enable research on important topics in developmental biology which are currently beyond the capabilities of biomedical science,
such as a study of the impact of differing environmental conditions, irrespective of genetic influence, on the development of pre-embryos.
-
American Society for Microbiology: Cindy L. Munro, "Genetic Technology and Scientific Integrity" (Chapter 10), in Francis L. Macrina (Virginia Commonwealth University),
Scientific Integrity
(3rd ed.) (American Society for Microbiology Press, March 2005, pf. 259) (http://www.asmpress.org/index.asp?downloadid=499): "Two [cloning] techniques (blastomere separation and somatic cell nuclear transfer)
are currently available in mammals;
blastomere separation has already been demonstrated with human cells. Human cloning is not a new topic for bioethical debate; the U.S. House of Representatives held hearings on the topic in 1978. However, it continues to be a difficult problem for those concerned with scientific integrity, as scientific and technological abilities outpace a consensus regarding appropriate use of the technologies. ...
In blastomere separation, a fertilized ovum is developed in vitro
to an early multicellular (up to 32-cell) stage. Each of the blastomeres is totipotent at this stage,
and careful division of the cell mass yields multiple cell masses,
each capable of developing into a genetically identical organism. For example, a 16-cell embryo can be divided to yield two 8-cell masses (resulting in identical twins) or four 4-cell masses (resulting in identical quadruplets).
Blastomere separation was first demonstrated with mouse embryos in 1970 and in cattle embryos in 1980.
In the context of infertility research, nonviable human embryos were duplicated using this technique in 1993; news reports generated a great deal of public debate regarding the ethics of the technology (18).
-
National Institutes of Health, "Cloning" (as of September 22, 2013),
(http://www.genome.gov/25020028).
The term
cloning
describes a number of different processes that can be used to produce
genetically identical copies of a biological entity. The copied material, which has the same genetic makeup as the original, is referred to as a clone. Researchers have cloned a wide range of biological materials, including genes, cells, tissues and even entire organisms.
Natural clones, also known as identical twins, occur in humans and other mammals.
These twins are
produced when a fertilized egg splits, creating two or more embryos that carry almost identical DNA. Identical twins have nearly the same genetic makeup as each other, but they are genetically different from either parent.
-
National Institutes of Health,
Report on Stem Cells
(June 17, 2001) (http://stemcells.nih.gov/info/scireport/appendixA.asp): "After fertilization, the zygote makes its way to the uterus, a journey that takes three to four days in mice and five to seven days in humans. As it travels, the zygote divides. The first cleavage produces two identical cells and then divides again to produce four cells.
If these cells separate, genetically identical
embryos
result, the basis of identical
twinning." (p. A-3)
-
Library of Congress: Judith A. Johnson, "Human Cloning", National Council for Science and the Environment, Congressional Research Service Report #RL313558, February 25, 2002, pp. 7, 11 (http://fpc.state.gov/documents/organization/9666.pdf):
"The NIH Panel
also identified areas of human embryo research it considered to be unacceptable, or to warrant additional review. It determined that
certain types of cloning [11]
without transfer to the uterus warranted additional review before the Panel could recommend whether the research should be federally funded.
"[Footnote 11]: These were
blastomere separation, where a two-to eight-cell embryo is treated causing the cells (blastomeres) to separate; and,
blastocyst division, in which an embryo at the more advanced blastocyst stage is split into two."
-
CRS Report for Congress: Irene Stith-Coleman, "Cloning: Where Do We Go From Here?", National Council for Science and the Environment, Congressional Research Service [Library of Congress] Report #97-335, April 23, 1998 (http://www.cnie.org/NLE/CRSreports/science/st-18.cfm): "In
Blastomere Separation, the outer coating, or zona pellucida, is removed from around a 2- to 8-cell embryo, then placed in a special solution that causes the cells, called blastomeres, to separate. Each cell can then be cultured individually, because
at this stage of embryo development, each cell is
totipotent, that is, it is undifferentiated and
can develop into an organism. After dividing a few times, each blastomere may develop into a smaller-than-normal
embryo
that can be transferred to the uterus.
"In
Blastocyst Division, also called induced
twinning, an embryo, at the blastocyst stage, a more advanced stage of development than the blastomere, is mechanically split into two. The two parts can be transferred to the uterus.
If both halves develop, then, at most,
one blastocyst gives rise to identical twins.
"In
Nuclear Transplantation, a nucleus is transferred
from each blastomere of a 4- to 8-cell or later-stage embryo
into the cytoplasm (cell contents other than nucleus) of an egg from which the genetic material has been removed (enucleated egg). To do this, the blastomere is placed beside an enucleated egg and their membranes are fused together artificially for example, with electrical pulses. The nucleus from the blastomere enters the egg cytoplasm and directs
development of the embryo.
"The Scottish scientists used a variant of the nuclear transplantation technique, where the nucleus that programmed the creation of Dolly was transferred from the adult sheep mammary cell, not an embryo. Researchers had thought that when cells became differentiated to do certain jobs in the body, they could not revert to the embryo stage. For example, they thought that a cell that became a liver cell remained a liver, but that belief was disproved by the Scottish scientists.
They were able to reprogram the genes in a mammary-gland cell to make it act like an undifferentiated embryo, which then developed into a sheep.
"(footnote #5: The information describing the
3 cloning techniques
was obtained from The
NIH Report of the Human Embryo Research Panel, September 1994.)"
Endnotes:
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