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Logical Biology 8 (2):52-54, 2008
OPEN LETTER
iPS CELL DISCOVERY AND HYPING

Evidence for Selection of Pre-existing Stem Cells Rather than Induction of iPS Cells
Shi V. Liu
(Received 2008-04-23; revised 2008-05-01; accepted 2008-05-10; published 2008-05-10*) HIGHLIGHT

Cell published a study in 2006 making a yet-unproven claim of directly reprogramming adult skin cells into
embryonic-like stem cells – the so called induced pluripotent stem (iPS) cells. Now this claim was
“proved” again in Cell but such proof is actually a disproof of the claim.
ABSTRACT
Yamanaka’s famous 2006 discovery of induced pluripotent stem (iPS) cells in Cell has been proven as invalid now (). Ignoring the solid criticisms, Jaenisch’s group recently published another paper in Cell to prove again the “direct reprogramming” and thus “induction” of iPS cells from differentiated cells. However, all the evidence for the “induction” of new stem cells is actually a reflection of selection of pre-existing stem cells. Thus, with this new and unequivocal evidence against the “induction” claim, Cell should retract all of its previous invalid iPS publications. However, Cell chooses once again to totally ignore valid criticism. KEY WORDS
Stem Cells, iPS, Induction, Reprogramming, Cloning, Embryonic, ES cells, Selection, Pre-existing, Hyping SIR, I am glad to read a formal disapproval against the “primary” iPS cells injected into the blastocysts the previous claims of proving induction of differentiated cells into iPS cells (Hanna et al., The generation of those “primary” iPS cells was 2008). This message actually echoes well what I achieved by infecting mouse embryonic fibroblasts have stated many times before (Liu, 2008a, b)[more (MEFs) carrying a constitutively expressed reverse tetracycline trans-activator driven by the ROSA26 Since this new study claimed of providing endogenous Nanog locus with the Dox-inducible reprogramming of terminally differentiated adult lentiviral vectors encoding Oct4, Sox2, c-Myc and cells to pluripotency” (Hanna et al., 2008), I paid very careful attention to identify evidence that can The generation of those “secondary” iPS cells was attempted with B cells at their different Unfortunately, the “definite” proof reported in differentiation stages: namely the “immature” pro- this new study actually came from a very indirect B and pre-B cells isolated from the bone marrow and unnecessarily complicated approach. The and the mature IgM+IgD+ B cells purified from the “directly reprogrammed” “secondary” iPS cells spleen of 8 week old adult chimeric mice. It was were obtained from adult tissues of chimeras by reported that, only after “optimizing” the “culture selecting those cells containing genetic marks for condition”, nonterminally differentiated B cells (the pluripotency which was originated somehow from above pro-B and pre-B cells) were “induced” into iPS CELL DISCOVERY AND HYPING
iPS cells called “iB-iPS cells” from the “immature” Secondly, the efficiency of “induction” is still B cells. It should be noticed that this optimization very low and at about the same level as reported in of the “culture condition” included not only adding the previous “inductions” if the calculation was IL-7, SCF, Fit-3L (for supporting B cell done in a different way. The authors claimed that development) and IL-4, anti-CD40 and LPS (for the efficiency is around 1 iPS cells from 27 to 34 proliferation of mature B cells) but also growing these B cells on OP9 bone marrow stromal cells However, the real efficiency should be based on how many B cells (non-transgenic and transgenic) Even so, the terminally differentiated B cells (the were plated rather than how many antibiotic- IgM+IgD+ B cells) were not induced into iPS cells resistant B cells were obtained and then used for the even under the above “optimized” culture continued “induction” process. The authors have condition, whether the reprogramming was done argued again on the possible requirement of a with the original 4 factors or even with all the 20 factors screened for nuclear reprogramming before. transcriptional factors for achieving a higher It turned out that, to achieve a “direct” efficiency. If that is the case, carrying out the “induction” of ultimate iPS cells across two differentiated cells, even more factors were added generations and through multiple steps would make and a “sensitizing” step was included to change the any refinement of the combination more difficult if cell identity (from B cells to macrophage-like cells). The adult spleen B cells derived from 10 Thirdly, adding not just more chemicals but also week-old chimeras were transduced with a some bone marrow cells into the “optimization” of retrovirus encoding C/EBPα and/or the IL7-Rα “culture condition” actually made the “induction” subunit and cultured on OP9 cells in the presence of process of iPS cells more complicated. The Dox which was needed for activating the four inclusion of extra materials and process not directly factors (Oct4, Sox2, Klf4 and c-Myc) already related with the assumed “direct reprogramming” contained in the transgenic B cells. “Induction” of does not help the clarification of any “induction” iPS cells, called B-iPS cells, was observed with mechanism but only makes it more elusive. using C/EBPα or C/EBPα plus IL7-Rα but not with Fourthly, it is worthy of notice that iPS cells “induced” are not the same as evidenced by, for Now my questions are: is this two-generation- clear examples, the heterogeneity in DH-JH spanning and multi-step-containing “generation” of rearrangements (Figure 2C of the report) and in the iPS cells really a “direct” reprogramming? Why methylation state of the Oct4 and Nanog promoters (Figure 4C of the report). Rather than explaining transcriptional reprogramming) require a cell this heterogeneity as a result of the operation of multiple fundamentally different reprogramming mechanisms I would argue the heterogeneity reprogramming with defined factors need the originated from the pre-existing diversity of cells
assistance from OP9 bone marrow stromal cells? containing the genetic marker for pluripotency Why didn’t the authors try a truly direct and one- (Nanog-GFP). Whatever is true, it clearly means step reprogramming, i.e., transducing all these that no simple unified reprogramming mechanism newly identified essential factors into the can explain the generation of diversified iPS cells. unmodified terminally differentiated B cells at one Fifthly and more decisively, the data clearly time and watch the reprogramming process in a shows a selection, not any induction, of some
pluripotency marker (Nanog-GFP)-containing cells In my view, this “definite” proof for “direct from the chimeras (see the Figure 6 of the report). reprogramming” just proves that there is no direct Thinking in this way, it would be interesting to see if any Nanog-GFP-negative iPS cells can be Firstly, whenever a truly direct reprogramming “induced” from those non-transgenic B cells. I was experiment, i.e., transducing reprogramming factors trying to find such data in this report but failed to into “resting” cells (or more correctly to say, cells held at non-proliferating conditions) was tried in Thus, the extra factors and steps added for the this study, it failed in “inducing” any iPS cells “induction” of iPS cells may be required for the whether the starting cells were “immature” non- activation of the reproduction program(s) of those terminally differentiated or “mature” terminally transgenic cells containing the pluripotency marker Nanog-GFP. This proliferation (to form visible colonies), selection (with the antibiotic resistance Logical Biol. 8 (2):52-54, 2008
Truthfinding Cyberpress )
iPS CELL DISCOVERY AND HYPING
selection) and identification (with the pluripotency This publication is sent to Cell and also Dr. marker Nanog-GFP) constituted a successful Jaenisch, specifically requesting their response. approach in selecting/identifying pre-existing cells containing pluripotency marker(s). However, this
success in selection/identification of stem cells or
pluripotent cells should never be confused with the
much
“inducing”/”generating”/”creating” stem cells or
pluripotent cells from differentiated cells (terminal
or nonterminal) (Liu, 2007).
So in summary I am confident that the data presented in this new study actually disproves the
claim of any “induction” of iPS cells from normal
(non-transgenic) differentiated cells. The altered
status of the claimed iPS cells as reported in this
new study was certainly not obtained in any one
step by any “direct” reprogramming.
References

Hanna, J., Markoulaki, S., Schorderet, P., Carey,
B.W., Beard, C., Wernig, M., Creyghton, M.P.,
Steine, E.J., Cassady, J.P., Foreman, R., et al.
(2008). Direct reprogramming of terminally
differentiated
pluripotency. Cell 133, 250-264. Liu, S.V. (2007). Invalidating the induction claim and adopting an activation mechanism for stem cells useful for regenerative medicine. Logical Biology 7, 96-99. Liu, S.V. (2008a). iPS cells: a more critical review. Stem Cells Dev Published advanced online doi: 101089/scd20080062. Liu, S.V. (2008b). Towards a balanced view on iPS Cells. Logical Biology 8, 32-38. * This manuscript was drafted first on April 23, 2008. Its final revision was finished on May 1, 2008 and then submitted to Cell on May 1, 2008. The revisions were based on informal but solid peer reviews which included opinions from editors of some mainstream journals. The final revision was agreed by all the experts consulted as “appropriate”. However, the submission was never responded by Cell, even after repeated email inquiries and phone calls. A protesting letter was sent to the editor-in-chief and other senior editors of Cell on May 8, 2008 but Cell still ignore this submission. Since Cell has done this truly irresponsible thing in the past towards my submissions, I decide to publish this manuscript as an OPEN LETTER. The publication here is the same as submitted to Cell except for the added highlight, abstract and keywords. Logical Biol. 8 (2):52-54, 2008
Truthfinding Cyberpress )

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