Human embryonic stem cells derived by somatic cell nuclear transfer

Abstract

Reprogramming somatic cells into pluripotent embryonic stem cells (ESCs) by somatic cell nuclear transfer (SCNT) has been envisioned as an approach for generating patient-matched nuclear transfer (NT)-ESCs for studies of disease mechanisms and for developing specific therapies. Past attempts to produce human NT-ESCs have failed secondary to early embryonic arrest of SCNT embryos. Here, we identified premature exit from meiosis in human oocytes and suboptimal activation as key factors that are responsible for these outcomes. Optimized SCNT approaches designed to circumvent these limitations allowed derivation of human NT-ESCs. When applied to premium quality human oocytes, NT-ESC lines were derived from as few as two oocytes. NT-ESCs displayed normal diploid karyotypes and inherited their nuclear genome exclusively from parental somatic cells. Gene expression and differentiation profiles in human NT-ESCs were similar to embryo-derived ESCs, suggesting efficient reprogramming of somatic cells to a pluripotent state.

Figure 1. Development of Monkey SCNT Embryos Reconstructed with Optimized Protocols

Although HVJ-E fusion was efficient, SCNT constructs required activation by electroporation for blastocyst formation. Elimination of ionomycin from the activation treatment further improved blastocyst development. I, ionomycin; DMAP, 6-DMAP; CM, compact morula. See also Tables S1 and S2.

Figure 2. SCNT Blastocyst Development Is Affected by Premature Cytoplast Activation

(A) Morphology of nuclear donor fetal fibroblasts before SCNT. (B) Poor-quality human SCNT blastocyst without distinct ICM produced with suboptimal protocols; note the presence of excluded cells. (C) Spindle-like structures detected when donor nuclei were introduced into intact MII oocytes, but not when introduction was conducted after enucleation. Arrowhead and arrow point at the maternal MII spindle and somatic cell spindle, respectively. (D) Somatic nuclear cell spindles were formed in cytoplasts when oocyte enucleation and fusion were conducted in the presence of caffeine. (E) Human SCNT blastocyst with prominent ICM (asterisk) produced after caffeine treatment. (F) NT-ESC colony with typical morphology derived from a caffeine-treated SCNT human blastocyst.

Figure 3. Development of Human SCNT Embryos and NT-ESC Derivation after Caffeine Treatment

(A) Improved blastocyst development of human SCNT embryos treated with caffeine. A total of 63 (five cycles) and 43 (three cycles) oocytes were utilized for SCNT without or with caffeine, respectively. Sixty (95.2%) and 42 (97.7%) oocytes survived after SCNT micromanipulations. (B) NT-ESCs were derived only from blastocysts produced with caffeine. See also Figures S1, S2 and S3.

Figure 4. Validation of Human SCNT with Nuclear Donor Cells Derived from a Leigh’s Disease Patient

(A) In vitro development of SCNT embryos produced with skin fibroblast cells from a Leigh’s disease patient and two different egg donors (egg donors B and C). Fifteen MII oocytes were retrieved from egg donor B, whereas only five oocytes were collected from donor C. SCNT blastocysts were generated from both oocyte cohorts. (B) NT-ESC derivation efficiency allowed isolation of one cell line per egg donor cycle. See also Figures S2 and S3.

Figure 5. Ovarian Stimulation and Human SCNT Outcomes

(A) Human SCNT development varied with the number of oocytes collected from each ovarian stimulation cycle. Cycles producing ten or fewer oocytes were associated with improved development of SCNT embryos. (B) The efficacy of NT-ESC derivation also positively correlated with fewer numbers of oocytes collected in the ovarian stimulation cycle. (C and D) SCNT embryo development from cycles treated with GnRH agonists or antagonists. Blastocyst development was higher for oocytes recovered from donors receiving a GnRH antagonist. NT-ESCs were derived only from oocytes recovered from donors receiving GnRH antagonist. See also Figures S2 and S3 and Table S3.

Figure 6. Genetic, Cytogenetic, and Pluripotency Analysis of Human NT-ESCs

(A) Nuclear DNA genotyping from four human NT-ESC lines (hESO-NT1, hESO-NT2, hESO-NT3, and hESO-NT4) determined by microsatellite parentage analysis. A total of 24 microsatellite markers were used for each analysis. The representative markers for D2S1333 and D4S413 loci demonstrate that the nuclear DNA in these cell lines was exclusively derived from the somatic HDF-f cell line. No contribution of oocyte nuclear DNA was detected. (B) mtDNA genotyping by Sanger sequencing demonstrated that all NT-ESC lines contain oocyte mtDNA. (C) Cytogenetic G-banding analysis confirmed that all NT-ESCs exhibited a normal 46XX karyotype (hESO-NT1 result is representative). (D) Human NT-ESCs expressed standard pluripotency markers detected by immunocytochemistry for antibodies against OCT4, NANOG, SOX2, SSEA-4, TRA-1–60, and TRA-1–81. Original magnification, ×200; Ph, phase contrast. Note that the upper-left image for hESO-NT1 is the same shown in Figure 2F. The upper-right image for hESO-7 is the same shown in Figure S5 (upper-right). (E) Histological analysis of teratoma tumors produced after injection of human NT-ESCs into SCID mice. An arrow and arrowhead in the top panel indicate intestinal-type epithelium with goblet cells (endoderm) and cartilage (mesodermal), respectively. An arrow and arrowhead in the lower panel depict neuroecto-dermal (ectoderm) and muscle (mesoderm) tissues, respectively. Original magnification, ×200. See also Figures S4 and S5 and Tables S4 and S5.

Figure 7. Microarray Expression Analysis of Human NT-ESCs

(A) Scatterplot analysis comparing expression profiles of human NT-ESCs (hESO-NT1) with IVF-derived ESC controls (hESO-7) and parental skin fibroblasts (HDF-f). Both IVF-ESCs and NT-ESCs displayed low transcriptional correlation to fibroblasts (left and middle) but were similar to each other (right). (B) Tree diagram analysis linking NT-ESCs to IVF-ESCs. See also Figure S6 and Tables S6, S7, and S8.