Disclaimer: This is Totally Untrue.


2.4.16 Entrance of the Tree of Life (STAP Cells)

2.4.16.1 Overview

STAP Cells were discovered, but destructed through traps on Nature around February 2014 CE. Humans reached the entrance of the Tree of Life through STAP Cells. However, the entrance was once closed.

2.4.16.2 Details

2.4.16.2.1 Outline

As mentioned before, the Japanese woman researcher Obokata Haruko learned at Vacanti's laboratory of Harvard University. She returned to Japan, worked at Japanese institute RIKEN and discovered the way to create Pluripotent cells leading to the Tree of Life in November 2011 CE.
Vacanti, RIKEN, and Harvard University applied Provisional International Patent on April 24, 2012 CE.
The monograph started to be submitted since April 2012 CE to the UK based world famous science journal "Nature" and so on. Since submission requires expert's experience, world famous scientist Dr. Sasai (笹井) joined the research in December 2012 CE.
Since provisional applications expire after 1 year, the Provisional International Patent Application was revised to be non-provisional International Patent Application on April 24, 2013 CE.
Nature examined the monographs (letters), requested some modifications and additional experiments, and accepted the monographs (letters) in December 2013 CE.

Thus the cells closely associated with Immortality and the Tree of Life were developed.
In summary, Differentiated cells (Adult cells) become STAP Cells with Pluripotency and without the ability of self-renewal through acid treatment, STAP Cells become STAP Stem Cells with the ability of self-renewal through LIF cultivation, STAP Cells become FI-SCs (FGF4 Induced Stem Cells) with possible Totipotency through FGF4 cultivation. (STAP Cells would change into 2 directions, STAP Stem Cells and FI-SCs.)

Nature and RIKEN announced discovery of Pluripotent cell creation and possible Totipotent cell creation on January 30, 2014 CE through 2 letters entitled
"Stimulus-triggered fate conversion of somatic cells into pluripotency"
and
"Bidirectional developmental potential in reprogrammed cells with acquired pluripotency."

However, accusations mainly about unimportant pictures promptly arose.
The 1st accusation would be PubPeer's following website with a vermilion picture. It pointed out that basic trace (Germline) of electrophoresis picture of a reference sample's DNA (Fig. 1i, Lane 3, Lymphocytes) in the 1st letter is erroneously absent and Lane 3 is edited. (The absence of basic trace (basic band) (Germline) on electrophoresis never occurs.)
* "PubPeer STAP Cells" https://pubpeer.com/publications/24476887
The 2nd accusation would be about shape of placenta in a picture. It claims that the circular shape of 2 placentas in 2 pictures (Fig. 1b right and Fig. 2g lower) of the 2nd letter are quite similar or identical for different placentas. (The circular placenta pictures' identity seems unclear because of their insufficient resolution.)
* "PubPeer Placenta Similarity in the 2nd Letter" https://pubpeer.com/publications/24476891
Other accusations followed.
The followers claimed that the authors fabricated the pictures and the whole letters are doubtful.

However, the things pointed out were not matters that would suggest intention of fabrication. If one intended to fabricate monographs, he/she never use clearly erroneous electrophoresis pictures or the same placenta picture. The things pointed out were rather matters that would suggest weird circumstances of the letters including Nature's referees (examiners). Yet, ordinary people couldn't figure out the meaning of accusations as well as the contents of the letters.
As far as the electrophoresis picture, it means that Nature's expert referees (examiners) might have intended to destroy Obokata's discovery, since Nature's benevolent expert referees never miss such childish errors particularly on such significant monographs after several revisions.
Regarding the placenta pictures, Fig. 1b right of the 2nd letter shows that STAP Cells possibly change into the placenta cells with green fluorescence, in contrast to Fig. 1a right's placenta, which wouldn't show green fluorescence, supposedly because ES Cells wouldn't change into the placenta cells. Fig. 2g lower shows that FI-SCs change into not only the placenta cells but also embryonic cells with green fluorescence. As mentioned before, Embryonic Stem Cells wouldn't change into the placenta cells and Trophoblast Stem Cells wouldn't change into the Embryonic Cells. On the premise that FI-SCs naturally possibly change into the placenta cells like STAP Cells and Trophoblast Stem Cells, Fig. 2g lower shows that FI-SCs possibly also change into Embryonic Cells. Then Fig. 2g lower picture is quite a derivative thing rather than an essential motif of the 2nd letter.
Anyway, the things pointed out wouldn't suggest intention of fabrication.

However, the denial campaign continued. Subsequently, Wakayama admitted the fabrication and survived.
(Sasai didn't admit the fabrication. Meanwhile, Sasai died and RIKEN disclaimed the Patent Application.)
STAP Cells and their involvement might be quite inconvenient for the power possibly favoring exclusive immortality and resurrection.

2.4.16.2.2 Background

2.4.16.2.2.1 Obokata's Discovery

As mentioned before, Obokata Haruko (小保方 晴子) (born 1984 CE) is a Japanese woman researcher in generative medicine. After graduating from Japanese university, she learned at Vacanti's laboratory of Harvard University since the summer of 2008 CE, continued seeking for the unknown Stem cells in animals through Kojima's Ultra-fine glass tubes at Vacanti's laboratory. Obokata improved the way to collect the small cells. Obokata returned to Japan in September 2009 CE.
Obokata examined the ability of the cells allying with Yamato Masayuki of Tokyo Women's Medical University (her ex-advisor at Japanese university) and the world famous mouse cloning expert Wakayama at Riken Center for Developmental Biology (CDN) in Kobe. (RIKEN (理研) is Japanese National Institute of Physical and Chemical Research.)
* "Riken CDB" http://www.cdb.riken.jp/en/
* "RIKEN in Wikipedia" http://en.wikipedia.org/wiki/RIKEN

On the other hand, Japanese woman researcher Dezawa (出澤) applied International Patent on Muse Cells on July 14, 2010 CE. According to Dezawa, pluripotent or multipotent cells named "Muse Cell" can be selected from commercially obtainable human mesenchymal cells such as bone marrow. (One cell among one thousand cells would be Muse Cell.) In addition, Dezawa presented efficient ways such as acidity treatment.
However, its pluripotency was unclear.
* "Muse Cell in Wikipedia" http://en.wikipedia.org/wiki/Muse_cell

As mentioned before, in relation to Human Embryogenesis, cells would be hierarchically categorized mainly as below from a viewpoint of differentiation and potency, and differentiation, as mentioned before possibly mostly regulated through "Beads-on-a String" Chromatin Fibre, was generally regarded irreversible.

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DNAs show some types in shape.

Shapes of DNA
from left to right
(1) DNA Double Helix, (2) Nucleosome, (3) 10 nm "Beads-on-a-String" Chromatin Fibre,
(4) 30 nm Chromatin Fibre, (5) 30 nm Chromatin Fibre (wide view),
(6) Active Chromosome, (7) Active Chromosome (wide view),
(8) Metaphase Chromosome, (9) Metaphase Chromosome (wide view)
*Attribution: http://en.wikipedia.org/wiki/File:Chromatin_Structures.png
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Initial Stages of Human Embryogenesis
*Attribution: http://en.wikipedia.org/wiki/File:HumanEmbryogenesis.svg

* "Human Embryogenesis in Wikipedia" http://en.wikipedia.org/wiki/Human_embryogenesis
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(Ⅰ) Fertilized Eggs (and 2-cell, 4-cell cleavage cells)
They have the ability conducting self-renewal and changing into (differentiating into) all types of cells including placenta cells. The ability changing into all types of cells including placenta cells is called "Totipotency."

(Ⅱ) Embryonic Stem Cells (ES Cells)
They can be obtained cultivating Inner Cell Mass (Embryoblast) of Blastocyst. They have the ability conducting self-renewal and changing into (differentiating into) all types of cells except placenta cells. The ability changing into all types of cells except placenta cells is called "Pluripotency."
* "Embryonic Stem Cells in Wikipedia" http://en.wikipedia.org/wiki/Embryonic_stem_cells


Blastocyst
*Attribution: https://en.wikipedia.org/wiki/File:Blastocyst_English.svg

(Ⅲ) Trophoblast Stem Cells (TS Cells)
Blastocyst consists of Inner Cell Mass (Embryoblast) (inside) and Trophoblast (outside). Inner Cell Mass becomes Embryonic Stem Cells, which wouldn't change into the placenta cells. Polar Trophectderm is a part of Trophoblast neighboring Inner Cell Mass. Polar Trophectderm changes into Trophoblast Stem Cells. In contrast to Embryonic Stem Cells, Trophoblast Stem Cells exclusively change into the placenta cells. (They could be in a sense, Unipotency mentioned below.)
* "Trophoblast in Wikipedia" https://en.wikipedia.org/wiki/Trophoblast
* "PubMed Trophoblast Stem Cells" http://www.ncbi.nlm.nih.gov/pubmed/21106963

(Ⅳ) Adult Stem Cells (Somatic Stem Cells)
They have the ability conducting self-renewal and changing into (differentiating into) some types, a few types of cells. The ability changing into some types or a few types of cells is called "Multipotency."
* "Adult Stem Cell in Wikipedia" http://en.wikipedia.org/wiki/Adult_stem_cell

(Ⅴ) Progenitor Cells
They have the ability conducting self-renewal and changing into (differentiating into) a specified type of cells. (e.g. muscle stem cells changing into muscle cells) The ability changing into a specified type of cells is called "Unipotency."

(Ⅵ) Differentiated Cells (Somatic Cells)
They neither have the ability conducting self-renewal nor changing into (differentiating into) another type of cells. (e.g. muscle cells)
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Obokata and her colleagues noted that Kojima's collection through Ultra-fine glass tubes might be creating Pluripotent Cells because of physical shocks rather than just collecting, and shocks or stress on cells could be essential. Then Obokata discovered an efficient way to create Pluripotent Cells from mouse differentiated cells (adult cells) through acidity treatment in April 2011 CE.

In this case, Obokata and her colleagues aimed to change differentiated cells into Pluripotent and self-renewal cells.
Then they had to show both
( i ) the fact that cells change into Pluripotent cells with the ability of self-renewal through Obokata's special method and
( ii ) the fact that the cells that Obokata picked out were differentiated cells before Obokata's special method.
As far as the latter fact ( ii ), Obokata selected Leukocyte cells, which have CD45-protein molecules on the surface, through Fluorescence Activated Cell Sorter being mediated by "CD45 antibodies with GFP (Green Fluorescent Protein)." (When CD45 antibodies with GFP stick CD45-protein molecules on the surface of the cells, GFP emits green fluorescence. Then cells with CD45-protein molecules (Leukocyte cells) can be selected through FACS.) Consequently, T-cells included in the selected Leukocyte cells showed TCR Rearrangement (T Cell Receptor Rearrangement). It means that the cell used was a differentiated cell.
In contrast, as far as the former fact ( i ), Obokata used Oct-4-GFP (Green Fluorescent Protein) transgene mice and acid. The cells of Oct-4-GFP transgene mice emit green fluorescence when Oct-4 genes work. Oct-4 gene is responsible for Embryonic Stem Cell's self-renewal. As mentioned above, Embryonic Stem Cells are Pluripotent Cells. Then when green fluorescence is emitted, it means Pluripotency and self-renewal.

Specifically, Obokata conducted as follows.
(A) Obokata selected Leukocyte cells (with CD45-protein molecules on the surface) from Oct-4-GFP transgene mouse spleens of 1-week old through Fluorescence Activated Cell Sorter being mediated by CD45 antibodies with GFP.
(B) The selected Leukocyte cells were stored in acidity (pH 5.7) at 37 °C for 25 minutes.
(C) Many cells died, but some 25% cells survived. The survived cells didn't have the ability of self-renewal.
(D) Then the survived cells were cultivated in LIF (Leukemia Inhibitory Factor) medium. Then 30% of the cultivated cells showed green fluorescence. It means that Oct-4 gene expressing (activating) self-renewal arose involving 25% x 30% = 8% cells. The green fluorescent cells were sorted through FACS.
(E) DNAs of the green fluorescent cells (Oct-4 expressed (activated)) were multiplied through PCR (Polymerase Chain Reaction) and analized with electrophoresis. The electrophoresis showed slightly small DNAs that prove TCR Rearrangement and experience of Differentiation of the cultivated green fluorescent cells.
(F) CD45 positive cells were treated in the same way (in low pH (pH 5.7) at 37°C for 25 minutes) and implanted in mice and teratomas were formed. (It supports Pluripotency of the cells.)
(G) CD45 positive cells treated in the same way (in low pH (pH 5.7) at 37°C for 25 minutes) were implanted in a blastocyst of a mouse (fertilized ovum) and the blastocyst was implanted in surrogate mouse's womb. The surrogate mouse gave birth to chimera mice. It means that the treated cell became Pluripotent.
(The cells that survived the acid treatment were named STAP Cells. (STAP: Stimulus-Triggered Acquisition of Pluripotency) The cells that survived the acid treatment, cultivated in LIF medium, and expressed OCT-4-GFP green fluorescence (self-renewal) (like Embryonic Stem Cells) were named STAP Stem Cells.)
(H) Other than that, the survived cells without the ability of self-renewal (STAP Cells) were cultivated in Trophoblast Stem-Cell medium with FGF4 (Fibroblast Growth Factor 4). Then the cultivated cells were implanted in a blastocyst of a mouse and the blastocyst was implanted in surrogate mouse's womb. In this case, green placentas of surrogate mice were seen. It suggests the possibility that STAP Cells became Totipotent cells through FGF4. The possible Totipotent cells were named FI-SCs (FGF4 Induced Stem Cells).

2.4.16.2.2.2 TCR Rearrangement and Immunity

Obokata's experiments require technical knowledge to be figured out.

As mentioned before, DNAs' sequences are generally completely replicated during Metotic cell divisions except Telomeres. However, the T-cells (T lymphocyte cells) including the Cytotoxic T-cell (Killer T-cell) and the B-cells of lymphocyte cells (B lymphocyte cells) show additional exceptional behaviors on DNA sequences, called V(D)J Recombination. (Lymphocyte cells are a kind of Leukocyte cells.) It means that a few DNAs of T-cells (T lymphocyte cells) and B-cells (B lymphocyte cells) spontaneously partly change DNA sequences, some DNA fragments are excluded, and the DNAs slightly vary in length.
Cytotoxic T-cells (Killer T-cells) and B-cells are responsible for destroying various hazardous foreign bodies (Antigens) such as viruses, bacterias, and toxic substances. (B-cells destroy Antigens drifting outside of (between) various cells of a mouse (or a human). However, when Antigens (viruses) enter into cells of a mouse (or a human), B-cells can't destroy them. B-cells wouldn't destroy Antigens lurking in cells. Then instead, Cytotoxic T-cells destroy the infected cells of a mouse (or a human) in which Antigens (viruses) are lurking.)

Regarding hazardous foreign bodies (Antigens), for example viruses, Viruses are capsule type entities containing DNAs or RNAs in the capsular structure that is far smaller than common cells. Once a virus enter a mouse (or a human), the virus stick a common cell, enter the cell, and release the DNA or RNA in the cell. The protein synthesis mechanism of the cell works on the DNA or RNA, proteins are produced, and replicated numerous viruses are created in the cell. The cell is filled with the replicated viruses, destroyed. The viruses will be released outside and enter other cells. Thus, cells are destroyed by viruses.

A Simplified Diagram of a Virus
*Attribution: https://en.wikipedia.org/wiki/File:Virus_stucture_simple.png

A Typical Virus Replication Cycle (from left to right)
*Attribution: https://en.wikipedia.org/wiki/File:HepC_replication.png

* "Introduction to Viruses in Wikipedia" https://en.wikipedia.org/wiki/Introduction_to_viruses

Since types of hazardous foreign bodies (Antigens) are like one million, millions of kinds of Cytotoxic T-cells and B-cells should be created in advance to prepare for unknown Antigens. (Cytotoxic T-cells are a kind of T-cells.)

T-Cell
*Attribution: http://en.wikipedia.org/wiki/File:Healthy_Human_T_Cell.jpg


B-Cell
*Attribution: http://en.wikipedia.org/wiki/File:Blausen_0624_Lymphocyte_B_cell.png

The original cells of T-cells and B-cells are Hematopoietic Stem Cells. Hematopoietic Stem Cells reside in bones along with bone marrow. Bone marrow is a fluid substance in bones. Bone marrow is a mixture of body fluids, fats, and various cells mostly from Hematopoietic Stem Cells.

Components of Bone Marrow
*Attribution: http://en.wikipedia.org/wiki/File:Gray72-en.svg

As partly seen below, Hematopoietic Stem Cells change into various cells such as Erythrocyte Cells (red blood cells), Myeloid Dendritic Cells (mDC) through Monocyte Cells, Natural Killer Cells, T-cells, B-cells, and so on. Myeloid Dendritic Cells (mDC) derived from Monocyte Cells would differentiate into Macrophage or Dendritic Cells, to be precise. (As seen below, B-cells are not the final form in differentiation (B-cells would differentiate into Plasma cells), while T-cells are the final form in differentiation.) (These differentiation mostly occurs in Bone Marrow, but T-cells are created mostly in thymus (then named T-cell) and the else of T-cells are created in the tonsils. Then T-cells move to the spleen. Other than that, B-cells created in Bone Marrow are immature B-cells (Naive B-cells without Immunoglobulins). Naive B-cells (Immature B-cells) move to the spleen and grow to be B-cells. B-cells and T-cells fight against hazardous foreign bodies mostly in the spleen.)

Differentiation of Hematopoietic Stem Cells
*Attribution: https://en.wikipedia.org/wiki/File:Hematopoiesis_simple.svg
* "Hematopoietic Stem Cell in Wikipedia" http://en.wikipedia.org/wiki/Hematopoietic_stem_cell
* "Thymus in Wikipedia" http://en.wikipedia.org/wiki/Thymus
* "Tonsil in Wikipedia" http://en.wikipedia.org/wiki/Tonsil


Main Organs of Vertebrate
*Attribution: https://commons.wikimedia.org/wiki/File:Anatomy_and_physiology_of_animals_main_organs_vertebrate_body.jpg

* "Spleen in Wikipedia" https://en.wikipedia.org/wiki/Spleen

Specifically, protruding parts of T-cells (including Cytotoxic T-cells) and B-cells vary like millions of kinds. The protruding parts of B-cells are called BCR (B-Cell Receptor). The protruding parts of T-cells are called TCR (T-Cell Receptor).

BCR is specifically Y-shaped protein, Immunoglobulin. The bottom of Y (Immunoglobulin) is connected with a B-cell. 2 protuberances of Y stick out.


Schematic View of a B-Cell with BCR

An Immunoglobulin consists of 4 polypeptide chains. (A peptide (chain) is a chain of amino acids.) 2 of the 4 chains are longer and called Heavy chains. The other 2 chains are shorter and called Light chains. Each polypeptide chain consists of some similar units of substances. The unit is called Immunoglobulin domain.
As shown below, a Heavy chain consists of 3 Constant Heavy parts (CH1, CH2, and CH3) and 1 Variable Heavy part (VH). One Constant Heavy part corresponds to an Immunoglobulin domain. One Variable Heavy part corresponds to an Immunoglobulin domain.
Likewise, a Light chain consists of 1 Constant Light part and 1 Variable Light part. One Constant Light part corresponds to an Immunoglobulin domain and One Variable Light part corresponds to an Immunoglobulin domain.

Schematic Diagram of Immunoglobulin
*Attribution: https://en.wikipedia.org/wiki/File:AntibodyChains.svg

Each Immunoglobulin Domain consists of 2 Beta Sheets. A polypeptide tends to be formed like sheets and a Beta Sheet is Sheet-shaped structure of polypeptide.

*Attribution: https://en.wikipedia.org/wiki/File:1gwe_antipar_betaSheet_both.png

There would be a variety of the sheets like below.

*Attribution: https://en.wikipedia.org/wiki/File:Beta-meander1.png

*Attribution: https://en.wikipedia.org/wiki/File:5CPAgood.png

Then an Immunoglobulin Domain consists of 2 Beta Sheets' sandwich layered structure, while a chain (composed of 4 or 2 domains) consists of a polypeptide.
When a Y-shaped Immunoglobulin consisting of 12 Immunoglobulin Domains is schematically indicated considering Immunoglobulin Domains and Beta Sheets, it would be like below.

* "Immunoglobulin Domain in Wikipedia" http://en.wikipedia.org/wiki/Immunoglobulin_domain
* "Beta Sheet in Wikipedia" http://en.wikipedia.org/wiki/Beta_sheet

The 2 tips of an Immunoglobulin stick to a part of a hazardous foreign body (a part of an Antigen), when the structure and property of the tips and the part of the Antigen fit. An Immunoglobulin stick to a part of of a hazardous foreign body (a part of an Antigen), inactivate the Antigen, and is called "Antibody." As mentioned before, since there would be like one million kinds of hazardous foreign bodies, millions kinds of Immunoglobulins and consequently millions kinds of properties at the tips of Immunoglobulins should be prepared.

*Attribution: https://en.wikipedia.org/wiki/File:Antibody.svg
* "Antibody in Wikipedia" https://en.wikipedia.org/wiki/Antibody

The mechanism creating over millions kinds of Immunoglobulins is V(D)J Recombination.
For example about human genetics, genes for Immunoglobulin Heavy Chains are located on the 14th chromosome (14th DNA). Specifically, some DNA sequences (gene segments) for Immunoglobulin Heavy Chains are scattered on the 14th DNA. There are some 107 DNA sequences (107 gene segments) scattered on the 14th DNA. 9 DNA sequences (9 gene segments) of the about 107 DNA sequences (107 gene segments) are responsible for Constant Heavy parts (CH1, CH2, and CH3). The 9 DNA sequences are called Cμ, Cδ, Cγ3, Cγ1, Cα1, Cγ2, Cγ4, Cε, and Cα2. (All Constant Heavy parts of a cell are created from one DNA sequence of the 9 DNA sequences.)
About 98 DNA sequences of the about 107 DNA sequences are responsible for Variable Heavy parts. The about 98 DNA sequences are categorized into 3 types, V gene segments, D gene segments, and J gene segments. V came from "Variable." D came from "Diversity." J from "Joining."
About 65 DNA segments of the about 98 DNA segments are V gene segments scattered on V's region, but the about 65 DNA segments a little differ each other. (They might be tentatively called here like V01, V02, ..., V65 being numbered from left to right in reference to the upper end (initial) horizontal DNA diagram line of the schematic diagram shown below.)
27 DNA segments of the about 98 DNA segments are D gene segments scattered on D's region, but 27 DNA segments a little differ each other. (They could be similarly like D01, ..., D27.) 6 DNA segments of the about 98 DNA segments are J gene segments scattered on J's region, but 6 DNA segments a little differ each other. (They could be like J01, ..., J06.)
On the other hand, a Variable Heavy part is created from one V gene segment, one D gene segment, and one J gene segment. Then for example, a Variable Heavy part might be created from V37, D19, and J04. For example, another Variable Heavy part might be created from V29, D13, and J05. Thus, the possible combinations about Variable Heavy part would be 65 x 27 x 6 = 10,530.
V, D, and J would be randomly adopted and a vast variety of proteins would be created.
The mechanism creating proteins corresponding to the adopted gene segments is DNA's cut-and-paste. V gene segments are scattered on V's region, D gene segments are scattered on D's region, and J gene segments are scattered on J's region as shown below. The first procedure is removal of DNA sequences between the adopted D and J (for example, between D19 and J04). As the second procedure, the adopted D and J (for example, D19 and J04) are connected. The third procedure is removal of DNA sequences between the adopted D and J (for example, between V37 and D19). As the fourth procedure, the adopted V and D (for example, V37 and D19) are connected. Consequently, the adopted DNA sequences (for example, V37, D19, and J04) are arranged in adjacency and the corresponding protein (a variety of Immunoglobulin) will be created. The adoption randomly occurs and diversity of Immunoglobulin is put into practice. (On the other hand, the most important 3 sites sticking to a part of hazardous foreign body are called Comlementarity Determining Regions (CDRs).)
Other than that, it would be noted that some DNA sequences are removed and the DNA is shortened.
Regarding Constant Heavy parts (CH1, CH2, and CH3), as mentioned above, 9 DNA sequences are responsible for them. Initially, the 9 DNA sequences are located from left to right as Cμ, Cδ, Cγ3, Cγ1, Cα1, Cγ2, Cγ4, Cε, and Cα2, to the right of J's region. Then, one DNA sequence is adopted like in Variable Heavy parts. As shown in the diagram below, the adopted DNA sequence for Constant Heavy parts will be located just after VDJ. The adopted DNA sequence for Constant Heavy parts designates Isotype of the Immunoglobulin.
Thus consequently, Heavy Chains theoretically provide 65 x 27 x 6 x 9 = 94,770 varieties.

*Attribution: http://en.wikipedia.org/wiki/File:VDJ_recombination.png

* "V(D)J Recombination in Wikipedia" http://en.wikipedia.org/wiki/V(D)J_recombination
* "Isotype (Immunology) in Wikipedia" http://en.wikipedia.org/wiki/Isotype_(immunology)

As far as Light Chains, as mentioned above, a Light Chain consists of one Variable Light part and one Constant Light part. Consequently, Light Chains theoretically roughly provide 40 x 5 x 2 = 400 varieties. (In contrast toVariable Heavy part, there is no D gene segments in reference to Variable Light part, and the Variable Light part is created from V gene segment and J gene segment. Then the DNA recombination in reference to Variable Light parts is called "VJ Recombination.")

Consequently, Immunoglobulins can theoretically provide roughly 94770 x 400 = 37,908,000 varieties.

TCR Complex is specifically six I-shaped proteins. The bottoms of Is are connected with a T-cell. The middle two long proteins among the six proteins are TCR (T-Cell Receptor). In most cases, TCR proteins are proteins that are named α chain and β chain. (Otherwise, TCR proteins are exceptionally γ and δ.) Then in most cases, the names of proteins are, from left to right, ε chain, δ chain, α chain, β chain, γ chain, and ε chain.

Schematic View of a T-Cell with TCR Complex

The 2 proteins of TCR are in most cases α chain and β chain. α chain consists of one Variable part and one Constant part. β chain consists of one Variable part and one Constant part.
Similar to Immunoglobulin's Variable Heavy part, β chain's Variable part comes from V, D, and J. Specifically, VDJ Recombination occurs on the DNA and a vast variety of β chains are created.
Regarding α chain, similar to Immunoglobulin's Variable Light part, α chain's Variable part comes from V and J. Specifically, VJ Recombination occurs on the DNA and a vast variety of α chains are created.
The tips of TCR with a vast variety bind to the targets.
DNA's length would be shortened through V(D)J Recombination about TCR such as α chain and β chain.

However, it should be noted that sexually reproducing organisms (mammals) are generally diploid, there are similar 2 DNAs from father and from mother, and V(D)J Recombination mentioned above occurs on one DNA. The other DNA would not be shortened.

Other than that, the other 4 proteins are called CD3. CD (Cluster of Differentiation) is a group of molecules such as proteins partly similar to Immunoglobulins. Molecules (including proteins) partly similar to Immunoglobulins are categorized and numbered in order of immunological conferential decisions. (Then "3" has no significant meaning.)

TCR Complex
*Attribution: https://en.wikipedia.org/wiki/File:TCRComplex.png

* "T Cell Receptor in Wikipedia" http://en.wikipedia.org/wiki/T_cell_receptor
* "CD3 (Immunology) in Wikipedia" http://en.wikipedia.org/wiki/CD3_(immunology)

In addition, T-cells are categorized into some variations depending on other molecules mostly associated with CD number on the surface. The variations are Helper T-cells (with CD4 protein), Cytotoxic T-cells (with CD8 protein), Memory T-cells (with CD45RO protein), Suppressor T-cells (with FOX protein), and so on.

Then immunity roughly proceeds as follows, while details are left out. A virus is a small infectious entity that can replicate only in living cells of other living things. When a mass of a type of viruses enter into a body of a mouse (or a human), the viruses would enter into cells of a mouse (or a human), parasitize the cells, proliferate in the cells, and parts of viruses would appear on the surface of the cells.
When Dendritic cells encounter the mass of the viruses or infected cells with parts of viruses on the surface, Dendritic cells partly destroy viruses and hold some fragments (parts) of the viruses. The Dendritic cells holding some fragments (parts of viruses) encounter Helper T-cells and Helper T-cells accept the fragments (parts) of the viruses. When the Helper T-cells judge that the parts should be attacked, Helper T-cells direct B-cells and Cytotoxic T-cells to be activated.

Dendritic Cell
*Attribution: https://commons.wikimedia.org/wiki/File:Dendritic_Cell_ZP.svg

Once B-cells are directed to be activated, B-cells differentiate into Plasma cells and Plasma cells produce Immunoglobulins corresponding to the parts of viruses. The Immunoglobulins destroy the parts of the viruses and inactivate the viruses drifting outside of cells of a mouse (or a human). (As mentioned above, B-cells and Immunoglobulins are responsible for hazardous foreign bodies (Antigens) drifting outside of cells of a mouse (or a human).)
Once Cytotoxic T-cells are directed to be activated, numerous activated Cytotoxic T-cells are created and infected cells with parts of viruses on the surface are destroyed. (As mentioned above, Cytotoxic T-cells are responsible for destroying infected cells in which Antigens (viruses) are lurking.)
*Precisely, BCRs of B-cells directly bind to parts of hazardous foreign bodies. In contrast, TCRs of T-cells require MHC's mediation.
* "Major Histocompatibility Complex in Wikipedia" https://en.wikipedia.org/wiki/Major_histocompatibility_complex

2.4.16.2.2.3 CD45 and FACS

Regarding CD45, it would be understood in reference to Cluster of Differentiation (CD) Numbers. As mentioned above, protein molecules on the surface of cells and partly similar to Immunoglobulins are categorized as CD numbers. The numbers correspond to the order of decisions in the immunological conferences. As partly seen below, CD45-protein molecules are seen on the surface of Leukocyte cells. Then CD45-protein molecules are generally called "Leukocyte Common Antigen (LCA)." (Though CD-protein molecules are not hazardous foreign bodies, they could be called "Antigens" for now.) ("CD45+" and "CD45 positive" mean that a CD45-protein molecule is present on the surface of the cell. "CD45+/CD3+" means that both CD45 and CD3-protein molecules are present on the surface of the cell. In contrast, "CD-" and "CD45 negative" mean that CD45-protein molecules are absent on the surface of the cell.)

*Attribution: https://en.wikipedia.org/wiki/File:Cluster_of_differentiation.svg
* "Cluster of Differentiation in Wikipedia" https://en.wikipedia.org/wiki/Cluster_of_differentiation
* "PTPRC in Wikipedia" https://en.wikipedia.org/wiki/PTPRC

Cells' selection through Fluorescence Activated Cell Sorter is a common way to select cells depending on CD types. In this method, Green Fluorescent Protein is used. For example, when antibodies corresponding to CD45-protein molecules are created and the antibodies are fused with Green Fluorescent Protein, the CD45 Antibodies with Green Fluorescence Protein stick CD45-protein molecules on the surface of the cell and emit Green Fluorescence in the presence of Ultra Violet (UV) light. CD45 Antibodies (with Green Fluorescent Protein) stick CD45-protein with extremely high accuracy.
Fluorescence Activated Cell Sorter is a device selecting cells depending on the presence/absence of each cell's fluorescence. The accuracy of FACS is quite reliable with over 99.5%.
Then cells with CD45-protein molecules are accurately collected with like over 99% accuracy.

2.4.16.2.3 Oct-4-GFP Transgene

Green Fluorescent Protein's gene can be used to detect specific gene's activation (expression) (specific gene's protein production).
As far as Obokata's experiment, activation (expression) (protein production) of Oct-4 gene was detected with Oct-4-GFP Transgene. Specifically, Oct-4-GFP Transgene mice were created. It is known that Oct-4 gene is responsible for embryonic stem cells' self-renewal. (Embryonic stem cells have the ability changing into various cells except placenta (Pluripotency) and self-renewal.) Then if a cell from a Oct-4-GFP Transgene mouse emits Green Fluorescence, it means that proteins associated with "Oct-4 gene and Green Fluorescence gene" are produced, and Pluripotent cell's self-renewal is being practiced. (GFP's gene is called a reporter gene.)
* "Oct-4 in Wikipedia" https://en.wikipedia.org/wiki/Oct-4
* "Reporter Gene in Wikipedia" https://en.wikipedia.org/wiki/Reporter_gene

The majority of the Leukocyte cells including B-cells, T-cells, and others die because of Obokata's acid treatment, but some 25% survives, while just the survived cells wouldn't show green fluorescence.
Subsequently, the survived cells are cultivated in LIF (Leukemia Inhibitory Factor) medium and some 30% of the cultivated cells start showing Green Fluorescence. Consequently, Oct-4 (responsible for embryonic stem cells' self-renewal) works on 8% of the original cells. It means that the cells became like Embryonic Stem Cells, which are self-renewal cells, through LIF medium.
* "Leukemia Inhibitory Factor in Wikipedia" https://en.wikipedia.org/wiki/Leukemia_inhibitory_factor

Additionally, there would be further 3 tests verifying the presence of Pluripotent Cells.
(1) Pluripotent Cells whould differentiate into various cells such as nerve cells and muscle cells through cultivation.
(2) Pluripotent Cells whould form teratoma when implanted in a mouse.
(3) Pluripotent Cells should result in a diploid chimera mouse when the cell is once implanted in a blastocyst of a mouse (fertilized ovum) and the blastocyst is implanted in a surrogate mouse's womb. (The surrogate mouse gives birth to a chimera mouse. The Oct-4-GFP diploid chimera mouse has green spots on its body. The green parts and the other parts have different DNAs. The mouse consists of 2 types of DNAs. Then it is called chimera.)
(Chimera mouse is the best test of the three to prove Pluripotency.)
*As mentioned before, diploid is common normal DNA number of animals. Tetraploid chimera mouse could be an additional pluripotent test, to be precise.

As results of the tests,
(1) Obokata's acid treated cells differentiated into various cells such as muscle cells and intestine cells.
(2) Obokata's acid treated cells formed teratoma when implanted in a mouse.
(3) The world famous expert Wakayama created diploid Chimera mice from Obokata's acid treated cells in November 2011 CE, and Obokata's acid treated cells' pluripotency was confirmed.

2.4.16.2.4 Electrophoresis, STAP Cell, and STAP Stem Cell

The other essential experiment is electrophoresis of the cells that were selected from mice spleens to prove TCR Rearrangement. As mentioned above in (D) and (E), Oct-4 expressed (activated) green fluorescent cells were sorted through FACS and DNAs of the green fluorescent cells (Oct-4 expressed (activated)) were multiplied through PCR (Polymerase Chain Reaction) and analized with electrophoresis. The electrophoresis showed slightly small DNAs that prove TCR Rearrangement. Since TCR Rearrangement occurs in T-cells and T-cells are Differentiated Cells (Somatic Cells) (Adult Cells), it means that once differentiated cells changed into Pluripotent self-renewal cells through Obokata's method.
Then the acid treated cells were named STAP Cells. (STAP: Stimulus-Triggered Acquisition of Pluripotency) The LIF cultivated cells with the ability of self-renewal were named STAP Stem Cells. (Additional ACTH (adrenocorticotropic hormone) in medium might be efficient.)
* "Adrenocirticotropic Hormone in Wikipedia" https://en.wikipedia.org/?title=Adrenocorticotropic_hormone

As far as DNA electrophoresis, an electric voltage is charged on a gel tray, multiplied DNAs are put on the negatively charged side, DNAs (with negative charge) move to the positively charged side in the gel. (Since Deoxyribo Nucleic Acid is an acid, it release H+ and the base DNA is negatively charged.) However, the velocities differ depending on molecular size (and electric charge) of the DNAs. Smaller DNAs move faster. Then DNAs are separated depending on molecular size (and electric charge). When T-cells' DNAs are the same in molecular size and electric charge, DNAs wouldn't be separated during electrophoresis. However, when TCR Rearrangement (V(D)J Recombination) occurred, DNAs are mixture of slightly randomly smaller DNAs and obscure stripes (a ladder) appear on the positively charged side.
However, as mentioned above, DNAs of mammals (sexually reproducing organisms) are diploid, TCR Rearrangement (random shortening) occurs only on one DNA of diploid, and the other DNAs wouldn't be shortened. Then clear trace (dense band) of DNA corresponding to the original DNA's molecular size should appear. The dense band (clear trace) of original DNA is called Germline (GL).
The following is Obokata's original electrophoresis picture. Gel 1 Lane 4 (CD45+cells) is a referential DNA (selected CD45 cells from spleens) and it correctly shows a dense Germline like ES cells (Lane 2) and obscure stripes from TCR Rearrangement. (Since ES cells are not differentiated, ES cells (Lane 2) wouldn't show obscure stripes.) The essential pictures here are Lane 5 and 6. Lane 5 and 6 are Oct-4 expressed and FACS sorted STAP Stem Cells' DNA. They similarly show a dense Germline and obscure stripes from TCR Rearrangement.

Obokata's Original Electrophoresis

2.4.16.2.5 FGF4 Induced Stem Cell

Other than that as mentioned above, the survived cells without the ability of self-renewal (STAP Cells) were cultivated in Trophoblast Stem-Cell medium with FGF4 (Fibroblast Growth Factor 4). Then the cultivated cells were implanted in a blastocyst of a mouse and the blastocyst was implanted in surrogate mouse's womb. In this case, green placentas of surrogate mice were seen. It suggests the possibility that STAP Cells became Totipotent cells through FGF4. The possible Totipotent cells were named FI-SCs (FGF4 Induced Stem Cells).
* "FGF4 in Wikipedia" https://en.wikipedia.org/wiki/FGF4

As mentioned above, in contrast to Embryonic Stem Cells, Trophoblast Stem Cells exclusively differentiate to the placenta cells. (Trophoblast wouldn't differentiate to other cells.) In contrast to Trophoblast Stem Cells, FI-SCs would possibly differentiate to both the placenta cells and other cells.

2.4.16.2.6 Application and Submission

Thus the cells closely associated with Immortality and the Tree of Life were developed.
In summary, Differentiated cells (Adult cells) become STAP Cells with Pluripotency and without the ability of self-renewal through acid treatment, STAP Cells become STAP Stem Cells with the ability of self-renewal through LIF cultivation, STAP Cells become FI-SCs with possible Totipotency through FGF4 cultivation. (STAP Cells would change into 2 directions, STAP Stem Cells and FI-SCs.)

Vacanti, RIKEN, and Harvard University applied Provisional International Patent on April 24, 2012 CE. The monographs (letters) started to be submitted since April 2012 CE to the UK based world famous science journal "Nature" and so on. Since submission requires expert's experience, world famous scientist Dr. Sasai (笹井) joined the research in December 2012 CE, Obokata and Wakayama studied surrounding matters. Since provisional applications expire after 1 year, the Provisional International Patent Application was revised to be non-provisional International Patent Application on April 24, 2013 CE. The applicants with potential ownership were the Brigham & Women's Hospital of Harvard University, RIKEN, and Tokyo Women's Medical University.

STAP Cell Patent Application

After several revisions and additional experiments, the world famous scientific journal Nature accepted the letters in December 2013 CE.

2.4.16.2.7 Announcement on January 30, 2014 CE and Accusations

Nature and RIKEN announced discovery of Pluripotent cell creation and possible Totipotent cell creation on January 30, 2014 CE through 2 letters entitled
"Stimulus-triggered fate conversion of somatic cells into pluripotency"
and
"Bidirectional developmental potential in reprogrammed cells with acquired pluripotency."
* "Nature 1st letter: Stimulus-triggered fate conversion of somatic cells into pluripotency" http://www.nature.com/nature/journal/v505/n7485/full/nature12968.html
* "Nature 2nd letter: Bidirectional developmental potential in reprogrammed cells with acquired pluripotency." http://www.nature.com/nature/journal/v505/n7485/full/nature12969.html

However, accusations mainly about unimportant pictures promptly arose.
The 1st accusation would be raised on February 4 in PubPeer's following website with a vermilion picture. It pointed out that basic trace (Germline) of electrophoresis picture of a reference sample's DNA (Fig. 1i, Lane 3, Lymphocytes) in the 1st letter is erroneously absent and Lane 3 is edited. (The absence of basic trace (basic band) (Germline) on electrophoresis never occurs.)
* "PubPeer STAP Cells" https://pubpeer.com/publications/24476887
The 2nd accusation would be about shape of placenta in a picture. It arose on February 13, 2014 CE stating that the circular shape of 2 placentas in 2 pictures (Fig. 1b right and Fig. 2g lower) of the 2nd letter are quite similar or identical for different placentas. (The circular placenta pictures' identity seems unclear because of their insufficient resolution.)
* "PubPeer Placenta Similarity in the 2nd Letter" https://pubpeer.com/publications/24476891
* "Placenta Similarity from goo.ne.jp" http://blogimg.goo.ne.jp/user_image/57/56/ bd9e7c2642ffe8d9179f7714ba6fe8e8.jpg
(The URL cited above is traced to "goo.ne.jp." "Goo" is a Japanese integrated website like ex-Rockefeller's "Yahoo.com" including Q&A and Blog website, by the way. ".jp" denotes Japan.)
Other accusations arose on February 14 in the article entitled "Alleged image manipulation" in the following website. (The following URL with ".jp" similarly originates in Japan.)
* "STAP Cell Blogspot.jp" http://stapcell.blogspot.jp/
They claimed that the authors fabricated the pictures and the whole letters are doubtful.
However, the things pointed out were not matters that would suggest intention of fabrication. If one intended to fabricate monographs, he/she never use clearly erroneous electrophoresis pictures or the same placenta picture. The things pointed out were rather matters that would suggest weird circumstances of the letters including Nature's referees (examiners). Yet, ordinary people couldn't figure out the meaning of accusations as well as the contents of the letters.
As far as the electrophoresis picture, it means that Nature's expert referees (examiners) might have intended to destroy Obokata's discovery, since Nature's benevolent expert referees never miss such childish errors particularly on such significant monographs after several revisions. (A possibility could be that Nature's referees might have insisted that Germline should be absent in referential Lymphocyte's electrophoresis pretending ignorance and RIKEN and Sasai might have compromised on removing the Germline to be accepted by Nature, since Lane 3 was merely a referential picture, while the true context is unclear.)
Regarding the placenta pictures, Fig. 1b right of the 2nd letter shows that STAP Cells possibly change into the placenta cells with green fluorescence, in contrast to Fig. 1a right's placenta, which wouldn't show green fluorescence, supposedly because ES Cells wouldn't change into the placenta cells. Fig. 2g lower shows that FI-SCs change into not only the placenta cells but also embryonic cells with green fluorescence. As mentioned above, Embryonic Stem Cells wouldn't change into the placenta cells and Trophoblast Stem Cells wouldn't change into the Embryonic Cells. On the premise that FI-SCs naturally possibly change into the placenta cells like STAP Cells and Trophoblast Stem Cells, Fig. 2g lower shows that FI-SCs possibly also change into Embryonic Cells. Then Fig. 2g lower picture is quite a derivative thing rather than an essential motif of the 2nd letter.
Anyway, the things pointed out wouldn't suggest intention of fabrication.
However, the denial campaign continued. Subsequently, Wakayama admitted the fabrication and survived.
(Sasai didn't admit the fabrication. Meanwhile, Sasai died in August 2014 CE, Tokyo Women's Medical University and RIKEN disclaimed the Patent Application in April 2015 CE.)
STAP Cells and their involvement could be quite inconvenient for the power possibly favoring exclusive immortality and resurrection, while an assassination cover-up and information control should be performed by an international political power.






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