Working Out the Various
Factors in iPS Cell Generation
by Jeremy Cherfas
The excitement generated by the announcement, in November
2007, that adult human cells could be reprogrammed into
stem cells, capable of differentiating into
any type of cell, shows no signs of dying down. The original
paper, from
Shinya
Yamanaka’s group at Kyoto University in Japan,
maintains its vice-like hold on the #1 spot despite a
fundamental problem with these induced pluripotent stem (iPS)
cells: one of the four triggers necessary to rejuvenate an adult
cell is a retrovirus called Myc, and re-activating the c-Myc
retrovirus gene unfortunately increases the likelihood of tumors
developing among the descendants of iPS cells. A follow-up paper
from essentially the same group, which addresses that
fundamental difficulty in the original paper, has now arrived at
#7.
Photo courtesy of Dr. Shinya Yamanaka, Kyoto
University.
Yamanaka’s team undertook a very thorough examination of
"family members" related to the four transcription factors that
reprogram adult cells, each time using the standard package plus
one homolog. For the first, Oct3/4, neither of the two closest
homologs was able to induce iPS cells. The second factor, Sox2, has
several homologs, of which six were tested. Sox1 did relatively
well, and two of the others induced some iPS cells. The third
factor is Klf4; three family members did induce iPS cells, but
inefficiently. c-Myc, the troublesome retroviral oncogene, has two
related proteins, and both N-Myc and L-Myc were able to induce
pluripotency.
So far so good. But as Yamanaka’s team reported,
"unexpectedly" a few stem-cell like colonies were obtained in the
absence of the Myc retrovirus. In the earlier studies, no iPS cells
had been obtained in the absence of Myc. The team noted that one
difference was in the timing of drugs to select the reprogrammed
cells; previously, they had started selection after 7 days, but in
the present study selection began after 14 days. As the team noted,
"This suggested that iPS cell generation without Myc is slower than
with Myc."
In a direct test of this hypothesis, the team started selection 7,
14, or 21 days after treating the cells with all four factors and
without Myc. Four factors gave positive colonies under all three
conditions, as expected, with substantially more iPS colonies when
selection was delayed. Three factors gave no colonies if selection
started at 7 days, again consistent with previous results, and
increasing numbers with selection at 14 and 21 days. Even more
interesting, the transformation was in one sense more efficient
without Myc, in that the percentage of positive colonies was higher
(even though the number of colonies was lower).
Injected into blastocysts (early-stage embryos), the three-factor
iPS cells gave rise to complete healthy adult mice with a high
proportion of tissues derived from the iPS cells. And these iPS
cells were associated with fewer tumors. Of 37 animals derived from
four-factor iPS cells, 6 died of tumors within 100 days of birth.
By contrast all of 26 animals derived from three-factor iPS cells
survived to 100 days. Of course these mice might have gone on to
develop tumors later in life, as the team acknowledged.
Other experiments showed that under various scenarios the three
factors, without Myc, were able to induce iPS cells at lower
efficiency and higher specificity. And human cells? They too can be
generated without using Myc. Colonies that resembled human
embryonic stem cells were generated at a low rate from skin cells,
and when these colonies were selected and expanded they generated
cells that expressed markers typical of embryonic stem cells. Those
cells could differentiate into at least three different cell types.
The paper’s conclusion makes it clear that there are
trade-offs. Without Myc there is a significantly lower risk of
tumors developing. The efficiency of generating iPS cells is,
however, considerably lower without Myc; in half the experiments
with human cells, it was not possible to generate iPS cells in the
absence of Myc.
Attention, in the meantime, has switched from the nuts and bolts of
generating iPS cells to the ways in which they can be put to use
and how they affect the ethical and practical landscape surrounding
the use of stem cells, embryonic and induced.
Parkinson’s
disease, heart disease, certain types of blindness and
neurological damage have all been candidates for iPS therapy.
Doubts about tumorigenicity remain, however, with some researchers
calling for renewed efforts to understand and use true embryonic
stem cells. A recent paper describing a method for creating iPS
cells free of vector and transgene sequences is attracting a lot of
interest, already registering in the Hot Papers Database (J. Yu,
et al., Science, 324[5928]: 797-801, 9 May 2009,
with 13 citations this period), and much of the attention seems to
be on using iPS cells to create populations of differentiated
cells, including cells specifically intended to exhibit certain
disease symptoms, that drug companies can use to screen new
compounds.
Dr. Jeremy Cherfas is Science Writer at Bioversity
International, Rome, Italy.
Biology
Top 10 Papers
Rank
Paper
Citations
This Period
(Jul-Aug 09)
Rank
Last Period
(May-Jun 09)
1
K. Takahashi, et al.,
"Induction of pluripotent
stem cells
from adult human fibroblasts by
defined factors,"
Cell, 131(5): 861-72,
30 November 2007. [Kyoto U.,
Japan; CREST, Kawaguchi, Japan;
Gladstone Inst. Cardio. Dis.,
San Francisco, CA] *243MG
88
1
2
Intl. HapMap Consortium (K.A.
Frazer, et al.), "A second
generation human haplotype map of
over 3.1 million SNPs,"
Nature, 449(7164): 854-61,
18 October 2007. [72 institutions
worldwide] *221LY
73
2
3
V. Cherezov, et al.,
"High-resolution crystal structure
of an engineered human
beta2-adrenergic G
protein-coupled receptor,"
Science, 318(5854):
1258-65, 23 November 2007. [Scripps
Res. Inst., La Jolla, CA; Stanford
U., CA] 233JG
42
6
4
N.J. Wilson, et al.,
"Development, cytokine profile and
function of human interleukin
17-producing helper T cells,"
Nature Immunol., 8(9):
950-7, September 2007.
[Schering-Plough Biopharma, Palo
Alto, CA; U. de Poitiers, France]
*202QN
37
†
5
E.V. Acosta-Rodriguez, et
al., "Interleukins
1ß and 6 but not
transforming growth
factor-ß are
essential for the differentiation
of interleukin 17-producing human T
helper cells," Nature
Immunol., 8(9): 942-9,
September 2007. [Inst. Res.
Biomed., Bellinzona, Switzerland]
*202QN
35
†
6
A. Grimson, et al.,
"MicroRNA targeting specificity in
mammals: Determinants beyond seed
pairing," Cell, 27(1):
91-105, 6 July 2007. [Howard Hughes
Med. Inst., MIT, Cambridge, MA;
Whitehead Inst., Cambridge, MA;
Rosetta Inpharmatics, Seattle, WA]
*190VK
34
†
7
M. Nakagawa, et al.,
"Generation of induced pluripotent
stem cells without Myc from mouse
and human fibroblasts," Nature
Biotech., 26(1): 101-6,
January 2008. [Kyoto U., Japan;
CREST, Kawaguchi, Japan; Gladstone
Inst., San Francisco, CA] *249IW
34
†
8
L. Zhou, et al., "IL-6
programs TH-17 cell
differentiation by promoting
sequential engagement of the IL-21
and IL-23 pathways," Nature
Immunol., 8(9): 967-74,
September 2007. [NYU Sch. Med., and
Howard Hughes Med. Inst., NY;
NHLBI, Bethesda, MD] *202QN
33
†
9
Intl. Consortium for SLEGEN, et
al., "Genome-wide association
scan in women with systemic lupus
erythematosus identifies
susceptibility variants in
ITGAM, PXK,
KIAA1542 and other loci,"
Nature Genetics, 40(2):
204-10, February 2008. [11
institutions worldwide] *256MJ
33
†
10
L. Yang, et al.,
“IL-21 and
TGF-ß are required
for differentiation of human
TH17 cells,"
Nature, 454(7202): 350-3,
17 July 2008. [Harvard Med.Sch.,
Boston, MA] *326ND