Understanding the genetic and environmental factors controlling pluripotency sheds incredible light on the basics of development not to mention defining sources for therapeutically useful cells and tissues. Additionally, the pathways controlling pluripotency in normal cells may become perturbed in certain abnormal biological conditions including germ cell-related tumors of the testes, ovary, and those occuring in other anatomical locations.
Park
IH, Arora N, Huo H, Maherali N, Ahfeldt T, Shimamura A, Lensch MW, Cowan
C, Hochedlinger K, and Daley GQ.
Disease-specific induced pluripotent stem cells.
Cell. 2008 Sep 5;134(5):877-86. (go
to PubMed)
Abstract:
Tissue culture of immortal cell strains from diseased patients is an invaluable resource for medical research but is largely limited to tumor cell lines or transformed derivatives of native tissues. Here we describe the generation of induced pluripotent stem (iPS) cells from patients with a variety of genetic diseases with either Mendelian or complex inheritance; these diseases include adenosine deaminase deficiency-related severe combined immunodeficiency (ADA-SCID), Shwachman-Bodian-Diamond syndrome (SBDS), Gaucher disease (GD) type III, Duchenne (DMD) and Becker muscular dystrophy (BMD), Parkinson disease (PD), Huntington disease (HD), juvenile-onset, type 1 diabetes mellitus (JDM), Down syndrome (DS)/trisomy 21, and the carrier state of Lesch-Nyhan syndrome. Such disease-specific stem cells offer an unprecedented opportunity to recapitulate both normal and pathologic human tissue formation in vitro, thereby enabling disease investigation and drug development.
(A) Two different,
primary fibroblast specimens, DS1 and DS2 from male patients with Down
syndrome (trisomy 21), were used to derive DS1-iPS4 and DS2-iPS10. Each
has a 47, XY+21 karyotype over several passages (G-banding analysis).
(B) Fibroblast (ADA and GBA) or bone marrow mesenchymal cells (SBDS) were
used to generate iPS cell lines. Mutated alleles identical to the original
specimens were verified by DNA sequencing. Adenosine deaminase deficiency
line ADA-iPS2 is a compound heterozygote: GGG to GAA double transition
in exon 7 of one allele (G216R substitution); the second allele is an
exon 10 frameshift deletion (-GAAGA). Shwachman-Bodian-Diamond syndrome
line SBDS-iPS8 is also a compound heterozygote: point mutations at the
IV2+2T > C intron 2 splice donor site and an IVS3-1G > A mutation
of the SBDS gene. GD-iPS3 (Gaucher disease type III): a 1226A > G point
mutation (N370S substitution) and a guanine insertion at nucleotide 84
of the cDNA (84GG). (C) Fibroblasts from patients diagnosed with either
Duchenne (DMD) or Becker type muscular dystrophy (BMD): DMD-iPS1 has a
deletion over exons 45–52 (multiplex PCR for the dystrophin gene).
We could not determine a deletion in BMD-iPS1 using two different multiplex
PCR sets though these assays do not cover the entire coding region. DMD2
is a patient control (exon 4 deletion). The control is genomic DNA from
a healthy volunteer. Huntington disease (HD) is caused by a trinucleotide
repeat expansion within the huntingtin locus. DNA sequencing shows that
HD-iPS1 has one normal (<35 repeats) and one expanded allele (72 repeats).
HD2 is a positive control from a second Huntington patient with one normal
and one expanded allele (54 repeats). The control is healthy volunteer
genomic DNA.
Lerou
PH, Yabuuchi A, Huo H,
Takeuchi A, Shea J, Cimini T, Ince TA, Ginsburg E, Racowsky C, and Daley
GQ.
Human embryonic stem cell derivation from poor-quality embryos.
Nat Biotechnol. 2008 (26): 212-4. (go
to PubMed)
Abstract:
During in vitro fertilization, embryos deemed clinically useless based on poor morphology are typically discarded. Here we demonstrate a statistical correlation between the developmental stage of such poor-quality embryos and the yield of human embryonic stem (hES) cell lines. Early-arrested or highly fragmented embryos only rarely yield cell lines, whereas those that have achieved blastocyst stage are a robust source of normal hES cells.
(a) Representative day-3 poor-quality embryos. From left to right, three-cell stage with asymmetry, 4-cell stage, 2-cell stage and 6-cell stage with fragmentation. Scale bar, 100 um. (b) Representative day-5 poor-quality embryos. Top row: expanding blastocyst with poor inner cells mass (ICM) development. Second row (left to right): two fragmented morulae, early blastocyst. Third row: three fragmented morulae. Bottom row (left to right): full blastocyst with poor ICM development, early blastocyst. Scale bar, 100 um. (c) Day-3 poor-quality human embryo arrested at the 6-cell stage with asymmetry and fragmentation. Scale bar, 100 um. (d) Colonies of the hES cell line CHB-1 derived from the embryo pictured in c. Scale bar, 200 um. (e) Fluorescence immunostaining of CHB-1 with anti-Oct3/4 (red) and anti-Tra-160 (green). Nuclei are stained blue with DAPI. Scale bar, 200 um. (f-k) Histopathology of tumors resulting from in vitro differentiation assay using hES cell lines derived from poor-quality embryos. Squamous epithelium (f), retinal epithelium (g), smooth muscle (h), bone (i), cartilage (j), columnar epithelium with goblet cells (k). Scale bars, 100 um. In addition to these differentiated regions, there were varying amounts of immature mesenchymal and neural tissue in each case (data not shown).
Park
IH, Zhao R, West JA, Yabuuchi A, Huo H, Ince TA, Lerou PH, Lensch MW,
and Daley GQ.
Reprogramming of human somatic cells to pluripotency with defined factors.
Nature. 2008 (451): 141-6.
(go
to PubMed)
Abstract:
Pluripotency pertains to the cells of early embryos that can generate all of the tissues in the organism. Embryonic stem cells are embryo-derived cell lines that retain pluripotency and represent invaluable tools for research into the mechanisms of tissue formation. Recently, murine fibroblasts have been reprogrammed directly to pluripotency by ectopic expression of four transcription factors (Oct4, Sox2, Klf4 and Myc) to yield induced pluripotent stem (iPS) cells. Using these same factors, we have derived iPS cells from fetal, neonatal and adult human primary cells, including dermal fibroblasts isolated from a skin biopsy of a healthy research subject. Human iPS cells resemble embryonic stem cells in morphology and gene expression and in the capacity to form teratomas in immune-deficient mice. These data demonstrate that defined factors can reprogramme human cells to pluripotency, and establish a method whereby patient-specific cells might be established in culture.
Multiple cultured human primary somatic cells yield iPS cells. a, iPS cells produced from five independent human primary cell lines form colonies with a similarly compact, ES-cell-like morphology in co-culture with mouse embryonic feeder fibroblasts (MEFs). b-e, As shown via immunohistochemistry (IHC), human iPS cell colonies express markers common to pluripotent cells, including alkaline phosphatase (AP), Tra-1-81, NANOG, OCT4, Tra-1-60, SSEA3 and SSEA4. 4,6-Diamidino-2-phenylindole (DAPI) staining indicates the total cell content per field. Fibroblasts surrounding human iPS colonies serve as internal negative controls for IHC staining. dH1f-iPS3-3 (b, from H1-OGN differentiated fibroblasts), MRC5-iPS2 (c, from MRC5 human fetal lung fibroblasts), BJ1-iPS1 (d, from neonatal foreskin fibroblasts), hFib2-iPS2 (e, dermal fibroblast from healthy adult male).
Kim
K, Ng K, Rugg-Gunn PJ, Shieh JH, Kirak O, Jaenisch R, Wakayama T, Moore
MA, Pedersen RA, and Daley GQ.
Recombination signatures distinguish embryonic stem cells derived by parthenogenesis
and somatic cell nuclear transfer.
Cell Stem Cell. 2007 (1): 346-52.
(go
to PubMed)
Abstract:
Parthenogenesis and somatic cell nuclear transfer (SCNT) are two methods for deriving embryonic stem (ES) cells that are genetically matched to the oocyte donor or somatic cell donor, respectively. Using genome-wide single nucleotide polymorphism (SNP) analysis, we demonstrate distinct signatures of genetic recombination that distinguish parthenogenetic ES cells from those generated by SCNT. We applied SNP analysis to the human ES cell line SCNT-hES-1, previously claimed to have been derived by SCNT, and present evidence that it represents a human parthenogenetic ES cell line. Genome-wide SNP analysis represents a means to validate the genetic provenance of an ES cell line.
SNP Genotype Data for SCNT-hES-1 and Three Representative Human ES Cell Lines: Genome-wide SNP mapping was performed using the GeneChip Human Mapping 500K SNP Array. (A) SCNT-hES-1. Genotyping data are depicted as in Figure 1, except that short p arm of the human chromosomes project superiorly, while long q arm projects inferiorly. Note pericentromeric regions of homozygosity for each chromosome. Conversion to homozygosity near telomeres is a reflection of the high frequency of double recombination in human chromosomes; (B) genotyping data are shown for three human ES cell lines (H9, BG01, and BG03) generated from fertilization embryos. The patterns of panheterozygosity were identical for all three lines (excepting the X chromosome data, which show homozygosity in the male line BG01); thus the data are presented as a composite. Orange blocks, homozygous (HOM) haplotypes; blue blocks, heterozygous (HET) haplotypes. (C) Heterozygosity of SNP markers plotted against SNP marker distance from the centromere for the four cell lines. Slope function is indicated. Error bars represent standard deviation.
Kim
K, Lerou P, Yabuuchi A, Lengerke C, Ng K, West J, Kirby A, Daly MJ, and
Daley GQ.
Histocompatible Embryonic Stem Cells by Parthenogenesis
Science. 2007 (315) 482: 482-6.
(go
to PubMed)
Abstract:
Genetically matched pluripotent embryonic stem (ES) cells generated via nuclear transfer or parthenogenesis (pES cells) are a potential source of histocompatible cells and tissues for transplantation. After parthenogenetic activation of murine oocytes and interruption of meiosis I or II, we isolated and genotyped pES cells and characterized those that carried the full complement of major histocompatibility complex (MHC) antigens of the oocyte donor. Differentiated tissues from these pES cells engrafted in immunocompetent MHC-matched mouse recipients, demonstrating that selected pES cells can serve as a source of histocompatible tissues for transplantation.
Genome-wide SNP genotyping of a representative clone of p(MII)ES, p(MI)ES, and panheterozygous p(MI)ES cells. (A) p(MII)ES cells, (B) p(MI)ES cells, (C) panheterozygous p(MI)ES cells. Left panels show genotypes for each chromosome, from centromere (cen, top) to telomere (tel, bottom), revealing blocks, or haplotypes, of markers. Green, homozygous C57BL/6 (B6) SNP; orange, homozygous CBA SNP; blue, heterozygous (HET) SNP. Right panels show the heterozygosity of SNP markers plotted against SNP marker distance from the centromere.
Chan
EM, Yates F, Boyer LF, Schlaeger TM, and Daley GQ.
Enhanced plating efficiency of trypsin-adapted human embryonic stem cells
is reversible and independent of trisomy 12/17.
Cloning Stem Cells. 2008 (10): 107-18. (go
to PubMed)
Abstract:
Human embryonic stem cells (hESCs) can be cultured abundantly and indefinitely, but are subject to accumulations of chromosomal aberrations. To preserve their genetic integrity, hESCs are commonly maintained as cell aggregates or clumps during passaging. However, clump passaging hinders large-scale culture and complicates the isolation of single cell clones. To facilitate the isolation of genetically modified clones of hESCs while preserving their genetic integrity, we employed trypsin single-cell passaging for brief periods before returning to clump passaging for long-term maintenance. We observed that accommodation to trypsin passage as single cells is an adaptive process where over three to four passages considerably increases the plating efficiency. However, trypsin passage was associated with abnormalities of chromosomes 12 and 17. Nevertheless, the high plating efficiency of trypsin passaged hESCs is a reversible phenotype, regardless of chromosomal abnormalities, suggesting that epigenetic events are responsible for the switch in phenotype.
The presence of trisomy 12/17 is not sufficient to confer a fast-growing or high plating efficiency phenotype to hESC lines. H9-collagenase and BGO1-collagenase cell lines of normal karyotype were adapted to trypsin for six and seven passages, respectively. H9-trypsin-adapted retained a normal karyotype, whereas BGO1-trypsinadapted acquired trisomy 12/17. H9-trypsin-adapted was then subsequently passaged with trypsin and collagenase in parallel for an additional 10 and 15 passages, respectively. Cells exposed to trypsin passaging (H9-trypsin-adapted P64/16) developed trisomy 12/17, whereas cells exposed to collagenase passaging (H9-recollagenase P69/6/15) were karyotypically normal. The cell line BGO1-trypsin-adapted P40/7, containing trisomy 12/17, was readapted to the collagenase method of clump passaging for another 14 passages and remained karyotypically abnormal. H9-collagenase was also passaged using a mild trypsin treatment (retains cells as small cell clumps) and after 15 passages was able to retain a normal karyotype (H9-trypsin/EDTA/clumps P42/15).