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Patient-Specific Embryonic Stem Cells Derived from Human SCNT Blastocysts
Woo Suk Hwang,1,2*Sung Il Roh,3Byeong Chun Lee,1Sung Keun Kang,1Dae Kee Kwon,1Sue Kim,1Sun Jong Kim,3Sun Woo Park,1Hee Sun Kwon,1Chang Kyu Lee,2Jung Bok Lee,3Jin Mee Kim,3Curie Ahn,4Sun Ha Paek,4Sang Sik Chang,5Jung Jin Koo,5Hyun Soo Yoon,6Jung Hye Hwang,6Youn Young Hwang,6Ye Soo Park,6Sun Kyung Oh,4Hee Sun Kim,4Jong Hyuk Park,7Shin Yong Moon,4Gerald Schatten7*
Patient-specific, immune-matched human embryonic stem cells(hESCs) are anticipated to be of great biomedical importancefor studies of disease and development and to advance clinicaldeliberations regarding stem cell transplantation. Eleven hESClines were established by somatic cell nuclear transfer (SCNT)of skin cells from patients with disease or injury into donatedoocytes. These lines, nuclear transfer (NT)–hESCs, grownon human feeders from the same NT donor or from geneticallyunrelated individuals, were established at high rates, regardlessof NT donor sex or age. NT-hESCs were pluripotent, chromosomallynormal, and matched the NT patient's DNA. The major histocompatibilitycomplex identity of each NT-hESC when compared to the patient'sown showed immunological compatibility, which is important foreventual transplantation. With the generation of these NT-hESCs,evaluations of genetic and epigenetic stability can be made.Additional work remains to be done regarding the developmentof reliable directed differentiation and the elimination ofremaining animal components. Before clinical use of these cellscan occur, preclinical evidence is required to prove that transplantationof differentiated NT-hESCs can be safe, effective, and tolerated.
1 College of Veterinary Medicine, Seoul National University, Seoul 151-742, Korea. 2 School of Agricultural Biotechnology, Seoul National University, Seoul 151-742, Korea. 3 Medical Research Center, MizMedi Hospital, Seoul 135-280, Korea. 4 College of Medicine, Seoul National University, Seoul 110-744, Korea. 5 Hanna Women's Clinic, Seoul 137-872, Korea. 6 School of Medicine, Hanyang University, Seoul 471-701, Korea. 7 Pittsburgh Development Center, Magee-Womens Research Institute, Department of Obstetrics, Gynecology, and Reproductive Sciences and Department of Cell Biology and Physiology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15213, USA.
* To whom correspondence should be addressed. E-mail: hwangws{at}snu.ac.kr (W.S.H.); gschatten{at}pdc.magee.edu (G.S.)
Many human injuries and diseases result from defects in a singlecell type. If defective cells could be replaced with appropriatestem cells, progenitor cells, or cells differentiated in vitro,and if immune rejection of transplanted cells could be avoided,it might be possible to treat disease and injury at the cellularlevel in the clinic (1). By generating hESCs from human NT blastocysts,in which the somatic cell nucleus comes from the individualpatient—a situation where the nuclear [though not mitochondrialDNA (mtDNA)] genome is identical to that of the NT donor—thepossibility of immune rejection might be eliminated if thesecells were to be used for human treatment (2, 3).
Recently, mouse models of severe combined immunodeficiency (SCID)(4) and Parkinson's disease (PD) (5) have been successfullytreated through the transplantation of autologous differentiatedmouse embryonic stem cells (mESCs) derived from NT blastocysts,a process also referred to as therapeutic cloning (6). In 2004,evidence was presented that a human NT-hESC line (NT-hESC-1)was derived by transferring the donor's cumulus cell nucleusinto her own enucleated oocyte (6); however, questions remainedas to whether the cell line had a parthenogenetic origin. Inaddition, it was not known whether NT-hESCs could be generatedfrom NT procedures using nuclei from males, prepubescent girls,or postmenopausal women, or when using cell components fromunrelated women. Finally, because NT-hESC-1 was grown on mousefeeders with likely xenograft contamination (7), the utilityof those cells is largely preclinical. In our study, patient-specificNT-hESCs were established reliably and efficiently (1 NT-hESCline per oocyte donation cycle) on human feeder cells and regardlessof somatic cell donor sex or age. In addition, some lines werederived without the animal products used during immunosurgery.Immunosurgery exposes the ESC precursors, the inner cell masscells (ICMs), through lysis of the blastocyst's outer trophectodermcells by sequential exposure to antibodies followed by complement;however, residual animal byproducts, including calf serum andenzymes, still remain. Furthermore, 10 of the 11 new cell lineswere generated from NT procedures using oocyte and somatic celldonors that were obtained from biologically unrelated individuals.
Oocyte donations by healthy women (with cell line -8 as theonly exception) and somatic cell donations by patients conformedto the regulations and law in the Republic of Korea, in accordancewith responsible institutional review board (IRB) review andoversight (8). Donors were fully aware of the scope of thisstudy and each signed an informed consent form. Both of theparents of children under 18 years old donating somatic cellswere similarly counseled, and each signed informed consent formson behalf of their child. Patients voluntarily donated oocytesand somatic cells for therapeutic cloning research and relevantapplications but not for reproductive cloning. Although expensesfor public transportation and injections administered by medicalpersonnel could have been provided, none of the donors requestedthis, and therefore no financial reimbursement in any form waspaid (9).
Recruited patients had a genetic immunodeficiency disease [congenitalhypogamma-globulinemia (CGH)], disorders caused by injury [spinalcord injury (SCI)], or another condition caused by complex autoimmunemechanisms [juvenile diabetes (JD)]. These three diseases areproposed to be treatable by single-cell-type transplantationwith hematopoietic stem cells (of mesodermal origin), motorneurons or neuroprogenitors (ectodermal origin), or ß-isletcells (endodermal origin), respectively. Patient-specific stemcells derived in this study are now expected to provide cellsin a disease state that can be used to understand disease progressionand assist in drug development. Because the stem cells generatedwith the use of patient cells are still likely to be defective,they probably cannot be directly used in cell transplantationto patients. In addition, before the cells can be used in theclinic, the biological properties of the patient-specific NT-hESCsmust be defined, reliable differentiation procedures must beestablished, and the cells must be free of contaminating undifferentiatedcells and potential pathogens.
Donor patients' fibroblasts were grown from skin biopsies (9).Individual cells were retrieved from the monolayer by trypsinization(9). Heterologous NTs were performed, in which donor somaticcell nuclei were transferred individually into enucleated oocytesfrom a biologically unrelated individual. However, for one cellline, NT-hESC-8, autologous NT was performed, in which the donor'sown fibroblast nuclei were transferred into her own enucleatedoocytes. Nine of the generated lines (and one of the unsuccessfulattempts) used donated oocytes from unrelated individuals (biologicalor otherwise), whereas another successful line and one unsuccessfulattempt used oocytes from a biologically unrelated family member.Details on oocyte and somatic cell donations and ovarian stimulationprotocols are described, and the Korean version and translationsby the Korean team into English of the informed consent formsare appended in (9). Enucleation, confirmation of the oocyte'sDNA removal, NT, fusion, and activation were performed as described(6).
Eleven NT-hESC lines were derived using somatic cells from patientswith SCI, JD, and CHG of both sexes and ranging from 2 to 56years old (Table 1). Figure 1 shows results from male NT-hESC-2(left columns) and female NT-hESC-3 (right columns), in whichstem cell colonies display characteristic cobblestone-like appearanceswith circumscribed borders (Fig. 1, A and B) and express hESCpluripotency markers, including alkaline phosphatase (AP) (Fig. 1, C and D),stage-specific embryonic antigen 4 (SSEA-4) (Fig. 1, G and H),SSEA-3 (fig. S1A), tumor rejection antigen 1-81(Tra-1-81) (Fig. 1, I and J), Tra-1-60 (fig. S1A), and octamer-4(Oct-4) (Fig. 1, K and L), but not SSEA-1 (negative control;Fig. 1, E and F). Normal male (Fig. 1M) and female (Fig. 1N)karyotypes are shown for NT-hESC-2 and NT-hESC-3, respectively.For complete data on NT-hESC-4 through -11, see fig. S1, B andC, and Table 2.
Fig. 1. hESC lines established from NT blastocysts using patient somatic cells (NT-hESCs) are pluripotent with normal karyotypes. The cell line for male NT-hESC-2 is shown in the left columns, and the cell line for female NT-hESC-3 is shown in the right columns. (A and B) Phase contrast imaging. (C to L) Pluripotency marker detections. Alkaline phosphatase(AP), (C) and (D); SSEA-1(notdetected), (E) and (F); SSEA-4, (G) and (H); TRA-1-81, (I) and (J); and Oct-4, (K) and (L) are shown. Magnification, x100; scale bars, 100 µm. (M and N) G-banded karyotyping (7) shows that NT-hESC-2 and -3 have normal XY and XX karyotypes, respectively.
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Table 2. Summary of patient-specific NT-hESC lines. ZF-blast, zona-free blastocyst; ImmS, immunosurgery; Plurip, pluripotent. The check marks denote pluripotency demonstrated by both EBs and teratomas. Normal karyotypes have been shown for each line. Lines derived from male patients are shown in blue; lines derived from female patients are in pink.
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Table 1. Establishment of patient-specific NT-hESCs.
DNA fingerprinting with human short tandem-repeat probes (Fig. 2and fig. S2, A to D) shows with high certainty that everyNT-hESC line derived here originated from the respective patientdonor and that these lines were not the result of enucleationfailures and subsequent parthenogenetic activation. In Fig. 2(red), isogenic analysis in the loci amelogenin, D5S818, andthe fibrinogen alpha chain gene (FGA) shows that the male lines-2 and -4 each precisely match the respective DNA fingerprintsof the male NT donors, just as the female line -3 is an identicalmatch with the female NT donor. The other male lines -5 and-9 to -12 also each match the respective male NT donors, justas the other female lines -6 to -8 each match the other femaleNT donors (fig. S2A; red are isogenic analyses in the amelogenin,D5S818, and FGA loci comparing NT-hESC-5 through NT-hESC-12,with their respective patient donor's DNA). DNA fingerprintingof donor oocytes was not performed because of their limitednumbers and their central importance for the NT procedure. Datashown in fig. S2B [black; isogenic analysis in loci D19S433,von Willebrand factor gene (vWA), thyroid peroxidase gene (TPOX),and D18S51], fig. S2C [green; isogenic analysis in loci D3S1358,tyrosine hydroxylase gene 1 (THO1), D13S317, D16S539, and D2S1338],and fig. S2D [blue; isogenic analysis in loci D8S1179, D21S11,D7S820, and c-fms proto-oncogene for CSF-1 receptor gene (CSF1PO)]further confirmed the identical matches of NT-hESCs with eachrespective donor. The statistical probability that these linesmay have been derived from another person is <4.1 x 10–16.
Fig. 2. DNA fingerprinting analysis of 3 of 11 NT-hESCs cell lines (-2 to -4) demonstrates genetic identities with donor patient nuclei (DNA fingerprinting of NT-hESC-5 through -12 are shown in fig. S2A). Isogenic analysis in loci amelogenin (chromosome location: X, p22.1 to 22.3; Y, p11.2); D5S818 (chromosome location 5p22 to 31); and FGA (chromosome location 4q28) is shown. The boxed numbers and corresponding peaks represent locations of polymorphisms for each short tandem-repeat marker at loci amelogenin (peak: X, Y); D5S818 (peak no. 9 to 13); and FGA (peak no. 18 to 26). Figure S2 provides additional DNA fingerprinting evidence.
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NT-hESCs have been efficiently established from a diverse groupof patients. In Table 1, the somatic cell donor's sex, age,and disorder or disease (Table 1B) are shown in the left columnsfor each of the 11 new NT-hESC lines (NT-hESC-2 to -12; Table 1A).Eighteen women donated 185 oocytes for these studies (Table 1C),of which 125 oocytes were donated by 10 women under 30years old (Table 1C, bottom row). On average, 10.2 oocytes weredonated during each assisted reproductive technology (ART) stimulationcycle, and an average of 12.5 oocytes were donated per cycleby women under 30 (Table 1C, bottom row). Success varied, withnine lines derived from single cycles (Table 1C). NT-hESC-2was established with five oocytes from a single cycle, but thederivation of NT-hESC-12 required 48 oocytes from five cycles(however, four of these donations were by women over 30; Table 1C).Lines NT-hESC-4 and -5 were established from a single cycle,as were NT-hESC-6 and -7 (Table 1C). The only line in whichthe oocytes and somatic cell were biologically related was NT-hESC-8,in which the donor's own somatic cell was transferred into herown oocytes. Two cycles each were required for NT-hESC-3 and-11 (Table 1C). Only two NT-hESC attempts [NT-hESC attempt I,with oocytes from two donors (one donation from a woman olderthan 30), and attempt II, with a single donor's oocytes] wereunsuccessful; i.e., NT constructs did not develop into blastocysts(Table 1A, bottom rows). Blastocyst development after NT occurredat high rates, with 129 fused NT constructs recovered from theoriginal 185 injected oocytes (69.7%; range from 33.3 to 95.5%;Table 1D). Thirty-one NT blastocysts (24.0% of the fused NToocytes; Table 1E) were generated. This rate was less than halfthe 60% in vitro fertilization (IVF) blastocyst developmentrate with gametes from infertility patients reported by ARTclinics (10).
Eleven NT-hESC lines were established from these 31 NT blastocysts(Table 1F; 35.4% average; range, 16.6 to 100%). 16.8 injectedoocytes or 11.7 NT-fused oocytes were required for each establishedNT-hESC line (range, 5 to 48 injected or 4 to 34 fused NT oocytes,respectively). NT-hESC lines were established from 40.9% ofNT blastocysts generated from oocytes donated by women under30 (Table 1F, bottom). For each NT-hESC line when the oocyteswere donated by women under 30, 13.8 injected oocytes or 10.0NT-fused oocytes were needed (Table 1F, bottom). Derivationrates for NT-hESC lines from NT blastocysts were similar tothose with fertilized blastocysts (11, 12).
Neither nuclear donor sex (table S1, 33.3% NT-hESC lines permale blastocyst versus 40.0% NT-hESC lines per female blastocyst)nor age (table S2) influenced cell-line establishment with statisticalsignificance. Oocyte donor age appears to be negatively correlatedwith success [oocytes donated by women older than 30 years wereless successful (table S3), with 40.9% NT-hESC lines per NTblastocyst for oocytes donated by women younger than 30 yearsversus 22.2% for oocyte donors older than 30 years]. Whereasthe limited sample size precludes findings of statistical significance,only two NT-hESC lines were established from the 60 oocytesdonated by women older than 30 (3.3% NT-hESCs per oocyte), andthe other nine lines were derived from the 125 oocytes donatedby younger women (7.2%). It is premature to conclude whethertechnical confounders, the natural variability of oocytes anddonor nuclei, or individual patient characteristics accountfor either very favorable outcomes (when some lines were establishedwith a few oocytes) or unsuccessful outcomes [when NT-hESCswere not generated (Table 1, attempts I and II)].
Stem cells are defined (13) by their ability to self-renew aswell as differentiate into somatic cells from all three embryonicgerm layers: ectoderm, mesoderm, and endoderm. Differentiationof 7 of the 11 new NT-hESC lines (NT-hESC-2 through -11) (Table 2)was analyzed both in teratomas (Fig. 3, A to X) and intoembryoid bodies (EBs) (Fig. 3, Y to e, and fig. S3), and theremaining four cell lines investigated so far differentiatedonly in embryoid bodies (Table 2). Each NT-hESC line differentiatedinto all germ layers. The convoluted surfaces characteristicof ectodermal lineages, including skin epithelium, and retinaland primitive neuroepithelium, are shown in Fig. 3, A, B, I, J, Q, and R.Muscle cell bundles, distinctive cartilage nodules,renal tubules, and bone matrix, all derived from mesoderm, areshown in Fig. 3, C, D, G, K, L, O, Q, S, T, W, and X. Endodermderivatives, including gastrointestinal and respiratory epithelia,are shown in Fig.3, E to H, M, N, P, U, V, and X. Figure 3, Y to e,demonstrates in embryoid bodies that each line differentiatesinto cells labeled by mono-specific lineage probes; for example,ectodermal cells [Fig. 3, Y to a; Y, NT-hESC-5; Z, NT-hESC-6;and a, NT-hESC-7; paired box gene (Pax 3/7), microtubule-associatedprotein 2 (MAP-2), and glial fibrillary acidic protein (GFAP)],mesodermal cells [Fig. 3, b to d; b, NT-hESC-8; c, NT-hESC-9;d, NT-hESC-10; atrial natriuretic peptide (ANP), CD34, and desmin,respectively], and endodermal cells [Fig. 3e, NT-hESC-11, myosinheavy chain (MHC)]. Figure S3 shows panels of immunohistochemicalmarkers specific for each lineage in every line analyzed todate (Table 2).
Fig. 3. Patient-specific human NT-hESCs differentiate into tissues from all three germ cell layers in vivo in teratomas (first three rows) and in vitro in EBs (bottom row). NT-hESC-2 (A to H), -3 (I to P), and -4 (Q to X) differentiated into all of the following somatic tissue types: skin [(A), (I), and (R)]; primitive neuroepithelium (B); striated muscle [(C) and (K)]; cartilage [(D), (L), and (T)]; renal tissues [(E) and (U)]; gastrointestinal epithelium [(F), (M), and (V)]; retina and primitive neuroepithelium [(J) and (Q)]; smooth muscle and respiratory epithelium [(G), (P), and (X)]; colon epithelium (H); mucosa gland (N); smooth muscle [(O) and (W)]; and bone (S). Immunohistochemical staining for EBs was performed for Pax3/7 (Y), MAP-2 (Z), GFAP (a), ANP (b), CD34 (c), desmin (d), and MHC (e). EBs also differentiated into all three germ layers expressing ectodermal [(Y), (Z), and (a)], mesodermal [(b) to (d)], and endodermal (e) marker genes. Figure S3 shows differentiation details for all NT-hESC lines. Magnification: x100, (A) to (R); x200, (S) to (Y). Scale bars, 100 µm.
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The discoveries of Wilmut et al. (14) in sheep cloning, togetherwith those of Thomson et al. (1) in deriving hESCs, have generatedconsiderable enthusiasm for regenerative cell transplantationbased on the establishment of patient-specific hESCs derivedfrom NT blastocysts generated from a patient's own nuclei. Thisstrategy, aimed at avoiding immune rejection through autologoustransplantation, is perhaps the strongest clinical rationalefor therapeutic cloning. By the same token, derivations of complexdisease-specific NT-hESCs may accelerate discoveries of diseasemechanisms. For cell transplantations, innovative treatmentsof murine SCID and PD models with the individual mouse's ownNT-mESCs are encouraging (4, 5). Human populations, unlike thoseof inbred mice, are genetically very diverse; consequently,conclusive proof of immune matching between the specific patientand her or his individualized NT-hESC line demands rigorousevidence. Toward this immune matching, MHC human leukocyte antigen(MHC HLA) isotypes of our stem cell lines are identical to thoseof the somatic cells from the corresponding patient donors (Table 3for lines NT-hESC-2 through -4; table S4 for lines -5 through-12). MHC-HLA matching is crucial for immunological toleranceduring organ donations, and in the absence of MHC-HLA matching,immunosuppressive medicines are required. This MHC-HLA matchingprovides additional evidence that parthenogenetic errors hadnot occurred. Each NT-hESC line matches the respective donor'sfor all HLA isotypes shown in Table 3 and table S4, suggestingthat transplanted NT-hESCs will be tolerated. Yet caution iswarranted in extrapolating from these in vitro data. Some histocompatibilityantigens traffic through mitochondria (15), and mitochondriain these NT-hESCs are likely to be of either oocyte or heteroplasmicorigin (except for autologous NT-hESC-8 and the original NT-hESC-1);thus, meticulous preclinical tolerance investigations in relevantpreclinical animal models are a prerequisite for any considerationof clinical experimentation.
Table 3. Identical matches of MHC-HLA isotypes among NT-hESC lines -2, -3, and -4 with donors. A, B, C, DRB, and DQB represent gene loci. M, male; F, female. Asterisks indicate genotyping analysis. See table S4 for MHC-HLA matches with NT-hESC-5 through -12.
MHC-1
MHC-II
HLA-A
HLA-B
HLA-C
HLA-DRB
HLA-DQB
Donor 2 (M)
A*01
B*37
Cw*06
DRB1*10
DQB1*0501
A*31
B*51
Cw*14
DRB1*14
DQB1*0502
NT-hESC-2
A*01
B*37
Cw*06
DRB1*10
DQB1*0501
A*31
B*51
Cw*14
DRB1*14
DQB1*0502
Donor 3 (F)
A*24
B*13
Cw*06
DRB1*04
DQB1*03
A*24
B*44
Cw*14
DRB1*11
DQB1*03
NT-hESC-3
A*24
B*13
Cw*06
DRB1*04
DQB1*03
A*24
B*44
Cw*14
DRB1*11
DQB1*03
Donor 4 (M)
A*02
B*60
Cw*03
DRB1*09
DQB1*0303
A*11
B*62
Cw*08
DRB1*15
DQB1*0602
NT-hESC-4
A*02
B*60
Cw*03
DRB1*09
DQB1*0303
A*11
B*62
Cw*08
DRB1*15
DQB1*0602
NT-hESCs were derived from 35.4% of the NT blastocysts (11 NT-hESClines/31 NT blastocysts). This rate is more than 10 times the3.3% reported earlier (6). In contrast, the rate of blastocystdevelopment remains at 24%. Direct derivations from either zona-freeor zona-enclosed intact blastocysts were superior to derivationsby immunosurgery (Table 2). Also, immunosurgery involves antibodiesand complement, so the elimination of this method avoids animalcontaminants, although alternatives for the animal enzymes andserum used in the fibroblast dissociation must now be perfected.
This 10-fold increase in NT-hESC derivation resulted from fiveprotocol improvements discussed here, combined with 10 thatwere previously reported (6). The five improvements that wedeveloped are as follows: (i) Human feeder cells, rather thanmurine ones, were established from the skin biopsy of donor2, obtained under local anesthesia, and grown in 10% fetal bovineserum, 1% nonessential amino acids, and 10 µg of penicillin-streptomycinper milliliter of culture medium at 37°C in a humidifiedatmosphere of 5% CO2 and 95% air. (ii) Donor nuclei were retrievedwith 0.25% trypsin-EDTA for 30 s at 37°C and monitored carefullyto avoid damage. (iii) Cumulus cell removal from the recipientoocytes demanded limited hyaluronidase exposure. (iv) DirectES derivations from NT blastocysts were performed, rather thanimmunosurgery (Table 2). (v) Scientist-specific micromanipulationimprovements were made during the most exacting steps of theoocyte's enucleation and during NT injection and fusion. Theprevious 10 important steps for NT blastocyst development arepresented in (6, 9). Neither autologous cytoplasmic matchesnor fructose now appears essential. These combined 15 stepsresult in an NT-hESC line established with 16.8 injected or11.7 NT-fused oocytes [fewer if donated by women younger than30 (13.8 injected or 10.0 fused oocytes)] (Table 1F), whichcompares favorably with 10.5 oocytes donated each cycle (12.5oocytes per cycle from women under 30 (Table 1C).
Our NT-hESC derivation rate is in line with some of the highestrates from IVF blastocysts (12). However, direct comparisonsneed to be tempered by the recognition that most IVF-hESC lineshave been derived from clinically discarded, frozen embryosfrom infertility patients (11, 12), whereas our studies reliedon only prime fresh oocytes donated by fertile women expresslyfor this research.
Here we have described the establishment of patient-specificNT-hESCs with high success rates (Table 2): Average rates indicatingthat each oocyte donation cycle leads to the establishment ofone patient-specific NT-hESC line. Furthermore, discoveriesof the mechanisms of complex and multifactorial diseases, alsocalled Research Cloning, are enabled because NT-hESC-9 is derivedfrom a patient with CGH, and NT-hESC-3 is derived from a patientwith JD. By extending NT-hESC procedures from previously autologous(6) to now heterologous NT, regardless of donor patient age(2 to 56 years old) or sex, these NT-hESCs can be evaluatedin vitro and after transplantation into appropriate animal modelsfor tolerance, efficacy, and safety.
NT-hESC derivation rates from NT-blastocysts increased morethan 10-fold over our previous results (6). Although the celllines in this study were derived on human feeder cells and werefound to be free of known contaminants, the method for dissociatingthe patient's skin biopsy and primary fibroblast culture includedfetal calf serum, trypsin, and collagenase (animal products).If preclinical results are encouraging, long before hESC linescould be used in the clinic, greater stringency is mandated,including methods to avoid xenoexposures. Furthermore, exactingdocumentation of the meticulous derivation, maintenance, anddifferentiation procedures would all need to be performed withincurrent good manufacturer practice facilities, so that regulatoryauthorities such as the Korean and/or U.S. Food and Drug Administrationcould evaluate investigational new drug applications. However,these rates of NT-hESC establishment, combined with a time frameof less than 1 year from skin biopsy and oocyte donation toNT-hESC establishment, might be clinically relevant if therapeuticcloning were shown to be of medical value.
Molecular deviations between animals developing after fertilizationversus those developing after reproductive cloning have beennoted. In particular, epigenetic aberrations have been discoveredin the genomic imprints of both cloned fetuses and offspring,as well as their placentas (2). Notwithstanding therapeuticinterest, learning whether the erasure, reestablishment, andstability of genomic imprints in these NT-hESCs compare withthose of IVF-derived hESCs (16) is essential. Other extranuclearor epigenetic influences include mitochondrial inheritance patternsin hESCs, let alone those in NT-hESCs, which are not understood.mtDNA heteroplasmy (17) could influence hESC stability or differentiation,as might homoplasmic oocyte mtDNA incompatibilities with thedonor nucleus. Furthermore, the cells' mitotic spindle poles,or centrosomes, which are contributed by the sperm during humanfertilization (18) and which, if imbalanced, have been shownto cause cancers (19, 20), might replicate or divide inaccurately,leading to aneuploidies. Consequently, learning whether somaticcentrosome transfer occurs during human NT, as it does duringbovine NT (21), is important. Inactivation of the X chromosome,which is typically random in female mammals, is skewed in clonedfemale mice (22, 23), causes recurrent spontaneous abortionsin some pregnant women (24), and could result in misexpressionsin NT-hESCs derived from women. Finally, the genomic stabilityof NT-hESCs, as well as their differentiation fidelity, includingaging and telomerase/telomere behavior, also require rigorousinvestigations.
The somatic cell's adaptation to in vitro conditions may predisposehuman NT embryos to cell culture proliferation, with negligiblepotentials for implantation and none for normal development.Neither NT embryonic development nor NT-hESC establishment ratesprovide any encouragement for dangerous human reproductive cloningattempts. Cloned animals have adverse pregnancy outcomes, soregardless of cruel hoaxes (25), scientific evidence shouldfurther discourage reckless notions regarding human reproductivecloning. Human SCNT was optimized from porcine SCNT proceduresin which 150 NT embryos were transferred for pregnancy establishment(26–28). Furthermore, in rhesus monkeys, 135 cloned embryostransferred into 25 surrogates using some of these improvedSCNT techniques (29) did not result in any pregnancies, althoughrhesus NT blastocysts developed and NT-ICMs were isolated.
Our work described here shows that stem cell lines can be generatedusing somatic cells from patients with disease and injury. Itmay also be possible to generate NT-hESC lines from patientswith diseases and disorders of unknown causes. For example,NT-hESCs derived from early-onset Alzheimer's disease or autismpatients might prove invaluable for mechanistic studies in vitroafter differentiation into neuroprogenitors (30, 31). In addition,biological insight gained through studying hESCs might findapplication to ART and assist in understanding genomic imprinting.The derivations of patient-specific NT-hESCs grown without animalcell co-culture may advance cell transplantation therapies aswell as aid in the discovery of human developmental processesand the causes of many complex diseases.
8. Before beginning any experiments, we obtained approval for this study from the IRB for Human Subjects Research and Ethics Committee at Hanyang University Hospital, Seoul, Korea, which was required by existing regulations that were in place up to 31 December 2004. IRB approvals are included in (9). Oocyte and/or somatic cell donors were counseled by two IRB members to ensure that they were fully aware of the scope of the investigation, and each donor signed informed consent forms. Both of the parents of children under 18 years old donating somatic cells were similarly counseled, and each signed informed consent forms on behalf of their child. On 1 January 2005, the Republic of Korea's new regulation entitled Bioethics and Biosafety Act—Act No. 7150, requiring governmental licensing of SCNT using human oocytes and subsequent derivation of NT-hESCs (Therapeutic Cloning), became effective. On 12 January 2005, we received governmental approval in accordance with this new stem cell law. This law also required IRB approval from the College of Veterinary Medicine, Seoul National University, which was granted on 25 January 2005. When our previous report on NT-hESCs appeared online on 12 February 2004 (6), we imposed a voluntary moratorium on new NT-hESC derivations. In September 2004, we announced that we were again performing SCNT and deriving NT-hESCs, under the auspices and oversight of the Hanyang University IRB for Human Subjects Research and Ethics Committee. All IRB documents are included in (9).
9. Materials, methods, and IRB documents are available as supporting material on Science Online.
31. O. Lindvall, R. McKay, Proc. Natl. Acad. Sci. U.S.A.100, 7430 (2003).[Free Full Text]
32. We thank foremost all donors. We also thank K. H. Han, J. H. Choi, J. T. Kang, S. G. Hong, and O. S. Kwon (Seoul National University); M. H. Kim, H. J. Jeong, E. K. Chun, and Y. J. Kim (MizMedi Hospital); and M. K. Koong and I. S. Kang (Samsung Cheil Hospital and Women's Healthcare Center) for assistance on NT-hESC culture; D. H. Chung (Seoul National University Hospital) for teratoma histopathology; J. Y. Kim and M. H. Park (Seoul National University Hospital) for HLA typing; and S. S. Yoo (Harvard Medical School) and the anonymous reviewers for their constructive critiques. All experiments were performed in Korea by Korean scientists, and all results were obtained in Korea using Korean equipment and Korean sponsorship. G.S and J.-H.P are grateful for the private philanthropy of the Magee-Womens Foundation, which supported their advisory roles in the analysis and for the interpretation and preparation for publication of these results obtained in Korea. No U.S. federal or Commonwealth of Pennsylvania funds were used in any aspect of this report. The authors are grateful for a graduate fellowship provided by the Ministry of Education, through BK21 program. This study was supported by grants from the Biodiscovery program of the Korean Ministry of Science and Technology to W.S.H. Until the formal establishment by the Republic of Korea of its National Center for Stem Cell Research, in which the previous NT-hESC line (-1) (6) and these new ones (-2 to -12) will be deposited and available for distribution, requests for cells and/or other materials should be addressed to W.S.H.
Correction of sickle cell disease by homologous recombination in embryonic stem cells.
L.-C. Wu, C.-W. Sun, T. M. Ryan, K. M. Pawlik, J. Ren, and T. M. Townes (2006)
Blood
108, 1183-1188
|Abstract »|Full Text »|PDF »
Embryonic Stem Cell-Derived Neuronally Committed Precursor Cells with Reduced Teratoma Formation After Transplantation into the Lesioned Adult Mouse Brain.
M. Dihne, C. Bernreuther, C. Hagel, K. O. Wesche, and M. Schachner (2006)
Stem Cells
24, 1458-1466
|Abstract »|Full Text »|PDF »
Role of Histone Acetylation in Reprogramming of Somatic Nuclei Following Nuclear Transfer.
1 NT-hESC line per oocyte donation cycle) on human feeder cells and regardless of somatic cell donor sex or age. In addition, some lines were derived without the animal products used during immunosurgery. Immunosurgery exposes the ESC precursors, the inner cell mass cells (ICMs), through lysis of the blastocyst's outer trophectoderm cells by sequential exposure to antibodies followed by complement; however, residual animal byproducts, including calf serum and enzymes, still remain. Furthermore, 10 of the 11 new cell lines were generated from NT procedures using oocyte and somatic cell donors that were obtained from biologically unrelated individuals. 
Oocytes = 185, 125
