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a Stemron Corporation, Inc., Gaithersburg, Maryland, USA;
b Reproductive Biology Association, Atlanta, Georgia, USA
Key Words. Stem cell • Homozygous • Metaphase • Oocyte
Correspondence:
Steve C. Huang, Ph.D., Stemron Corporation, Inc., 20 Firstfield Road, Suite 100, Gaithersburg, Maryland 20878, USA. Telephone: 240-631-7721; Fax: 240-631-1918; e-mail: shuang{at}stemron.com
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ABSTRACT |
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INTRODUCTION |
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Immunogenicity of a stem cell derivative depends on the expression of the major histocompatibility complex (MHC) genes that are highly polymorphic for each antigen. This polymorphism is further amplified by heterozygosity, as in a population with n1, n2, and n3 defined antigen specificities for HLA-A, -B, and -DR, respectively, resulting in theoretical phenotypes being as many as [n1*(n1+1)/2]* [n2*(n2+1)/2]*[n3*(n3+1)/2]* [1]. As observed in the National Marrow Donor Program, 302,867 HLA-A, -B, and -DR phenotypes were distinguished from 406,503 HLA-A, -B, and -DR-typed donors, revealing that on average each HLA-A, -B, and -DR phenotype observed was only presented 1.3 times [1]. Although limiting the immune response by eliminating MHC genes has been theorized as a potential method for reducing immunogenicity of transplanted cells, mouse MHC class-I and class-II-deficient skin grafts are still rejected, and it is proposed that "self" MHC molecules are desired for tolerance [2]. The number of phenotypes a graft can fully match depends on its HLA-A, -B, and -DR gene diversity. With n1, n2, and n3 different polymorphic alleles for -A, -B, and -DR, respectively, in the general population, an allograft heterozygous for all -A, -B, and -DR will match only one phenotype; an A-homozygous, B- and DR-heterozygous graft matches as many as n1 phenotypes; an A- and B-homozygous and DR- heterozygous graft matches n1*n2 different phenotypes; and an A-, B-, DR-homozygous graft matches n1*n2*n3 different phenotypes.
In contrast to ES, EG, cord blood, and adult stem cells, stem cells derived from metaphase II oocytes, as those derived from parthenogenesis, are either uniformly homozygous or include minimal crossover-associated heterozygosity. A graft derived from such "homozygous stem cells," presenting as few as three or four antigens, matches a much wider range of phenotypes than a heterozygous graft, and since some HLA haplotypes have a higher distribution in the population, for example, in North America the 15 most common HLA-A, -B, and -DR haplotypes match 21.2% Caucasian Americans [1], the use of homozygous stem cells renders the plausibility of creating a bank of stem cells covering most phenotypes in the general population. This bank could be utilized to create allogenic cell-based therapies for a variety of disease states, such as Parkinson’s, Alzheimer’s, and diabetes.
Although establishment of stem cell lines from homozygous materials has been studied and well demonstrated since 1983 in mouse via parthenogenetic activation [3], the pluripotency of the cells and the efficacy of their derivatives has been poorly explored. While some studies implied partial differentiation defect resulting from parthenogenetic stem cells [4], others displayed unrestricted lineage differentiation in chimeras [5]. However, differentiation potential comparable with heterozygous cells can be inferred favorably for homozygous stem cells from observations such as A) homozygosity is associated with fully differentiated tissue components (brain, skin, cardiac muscle, and bone marrow) in human ovarian teratomas [6]; B) laboratory inbred mice are homozygous for almost all genetic loci, and C) a human chimerism displays virtually 100% composition of the blood leukocytes derived from a parthenogenetically activated oocyte [7].
In this study, aiming at exploring the potential of using homozygous stem cells for cell-based therapies, we established mouse homozygous stem cell lines from metaphase II oocytes and characterized their "stemness" by examining the expression of stage-specific embryonal antigens (SSEAs), Oct4, and telomerase, the tissue components in teratomatous growth, and by assessing their ability to be induced in vitro by specific growth factors for ectoderm, endoderm, and mesoderm cell types. In addition, we assessed the feasibility of applying homozygous stem cell technology in humans. Superovulated metaphase II oocytes from a female human subject were subjected to mechanical and chemical manipulations that simulated biochemical responses in fertilization. We assessed these unfertilized oocytes for their potential to be activated in vitro, to progress to form blastocysts, and to produce a viable inner cell mass for stem cell development.
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MATERIALS AND METHODS |
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Characterization of Mouse Homozygous Stem Cell Lines
Three mouse metaphase II oocyte-derived cell lines (L5, L10, and LC57) at passage 10 were subjected to karyotype and genotype analyses, and characterized for their expression of alkaline phosphatase, SSEAs, Oct4, and telomerase. For karyotyping, metaphase chromosomes were prepared by exposing cells to 60 ng/ml colcemid (Sigma) overnight, followed by 75 mM KCl hypotonic solution treatment. The spreads were stained with Giemsa (Sigma), and the chromosome number of each spread was assessed with a Leica DM IRB inverted research microscope (McBain Instruments; Chatsworth, CA; http://www.mcbaininstruments.com). To confirm homozygosity in established lines, polymerase chain reaction (PCR)-based haplotype analyses using the microsatellite markers D2mit42 and D17mit232 (Research Genetics; Huntsville, AL; http://www.resgen.com) and an H2-I-A-marker (5'-GCT CCT CAA GCG ACT GTG TTC and 5'-CAC GGT TGA CGA AGA AGC TGG) with Mae III (Roche Biochemicals; Indianapolis, IN; http://www.roche.com) restriction digestions of PCR products were performed to distinguish B and D haplotypes. Fast Red substrate-chromogen (DAKO; Carpinteria, CA; http://www.dako.com) was used for alkaline phosphatase activity detection; anti-SSEA-1 (MC480), anti-SSEA-3 (MC631), and anti-SSEA-4 (MC813-70) (Developmental Studies Hybridoma Bank; University of Iowa; Iowa City, IA; http://www.uiowa.edu/
dshbwww), and fluorescein isothiocyanate (FITC)-conjugated secondary antibodies (Jackson ImmunoResearch Laboratories; West Grove, PA; http://www.jacksonimmuno.com) were used to detect SSEAs. Reverse transcription (RT)-PCR assays employed primers 5' GGC GTT CTC TTT GCA AAG GTG TTC/5' CTC GAA CCA CAT CCT TCT CT and 5' GAT GGC ATA CTG TGG ACC TCA G/5' CAG ATG GTG GTC (Oct4 was used in RT-PCR). Telomerase activities in the cell extracts were assayed using the TRAPeze telomerase detection kit (Intergen; Purchase, NY; http://www.intergen.com) in accordance with the manufacturer’s protocol.
In Vivo Differentiation of Mouse Homozygous Stem Cell Lines
Approximately one third of a colony of each established cell line (L5, L10, and LC57) at passage 10 was transplanted under the kidney capsule of an anesthetized syngeneic mice for teratomatous tissue formation. Six to eight weeks after transplantation, the resulting tissue growth was examined histologically.
In Vitro Differentiation of Mouse Homozygous Stem Cell Lines
Endodermal Differentiation
For endodermal lineage differentiation, cells from the established line LC57 were first cultured in suspension dishes (171099, 35 x 10 mm; Nalge Nunc International; Rochester, NY; http://www.nalgenunc.com) for 5 days in LIF-free stem cell medium to form embryoid bodies (EBs). The EBs were then transferred to a 0.1% collagen type I-coated (Sigma) 24-well plate (Corning; http://www.corning.com) in LIF-free stem cell medium containing 100 ng/ml acidic fibroblast growth factor (aFGF; Sigma) and cultured for 3 days. The medium was then replaced with LIF-free stem cell medium containing 20 ng/ml hepatic growth factor (HGF; Sigma) and incubated for 6 days. The differentiated cells were analyzed for the expression of endoderm-specific genes by RT-PCR assays employing the following primers: 5'-CTC ACC ACA GAT GAG AAG/5'-GGC TGA GTC TCT CAA TTC (transthyretin [TTR]); 5'-TCG TAT TCC AAC AGG AGG/5'-AGG CTT TTG CTT CAC CAG (
-fetoprotein [AFP]); and 5'-AAT GGA AGA AGC CAT TCG AT/5'-AAG ACT GTA GCT GCT GCA GC (-1-anti-trypsin [AAT]). The expression of ß-actin was assayed as a positive control using 5'-TTC CTT CTT GGG TAT GGA AT/5'-GAG CAA TGA TCT TGA TCT TC primers. For comparing the levels of gene expression in different stages, all RT-PCRs were performed in the same manner: 1 µg of total RNA extracted from a different differentiation stage was converted to cDNA in a 20-µl reaction; 1 µl of cDNA was used for PCR; and 10 µl of the amplification were used in gel electrophoresis.
Ectodermal Differentiation For differentiation of tyrosine-hydroxylase-positive neuronal cells, EBs from line LC57 were selected for nestin-positive cells by culturing in serum-free insulin/transferrin/selenium/fibronectin (ITSFn) medium: 50% DMEM, and 50% F12 (Life Technologies) supplemented with 5 µg/ml insulin, 30 nM selenium chloride, and 5 µg/ml fibronectin (Sigma). After 6-10 days of nestin selection, cells were dissociated by trypsin/EDTA and plated on 15-µg/ml poly-L-ornithine and 1-µg/ml fibronectin (Sigma) precoated 24-well plates at a concentration of 1.5-2 x 105 cells cm-2 in N2 medium: 50% DMEM and 50% F12 supplemented with N2 supplement (Life Technologies), 20 µg/ml insulin, 1 µg/ml laminin (Sigma), 10 ng/ml basic (b)FGF (R&D Systems; Minneapolis, MN; http://www.rndsystems.com), 500 ng/ml murine N-terminal fragment of sonic hedgehog, and 100 ng/ml murine FGF8 isoform b (R&D Systems). The medium was changed every 2 days. After 6 days of culture, bFGF was removed from the medium, and cells were further cultured for 6-15 days. To monitor nestin-positive cell selection, RT-PCR assay was performed using 5'-GGA GTG TCG CTT AGA GGT GC/5'-TCC AGA AAG CCA AGAG GAA GC primers. Primary antibodies, mouse anti-nestin (Chemicon), and rabbit anti-tyrosine hydroxylase (Pel Freez; Rogers, AR; http://www.pel-freez.com) were used for immunocytochemical detection of nestin and tyrosine hydroxylase, respectively; the secondary antibodies and substrate for detection from Envison Doublestain System (DAKO) were used according to manufacturer’s instructions.
Mesodermal Differentiation Cells from the established line L10 at passage 10 were subjected to hematopoietic differentiation by first forming EBs in the presence of 4.5 x 10-4 M monothioglycerol (Sigma). Approximately 30-40 EBs were transferred to a 35-mm dish containing 3 ml methylcellulose-based hematopoietic cell differentiation medium M3434 (StemCell) and incubated at 37oC and 5% CO2 for 10 days, followed by further culture on 35-mm dishes in Iscove’s modified Dulbecco’s medium (IMDM) supplemented with 10% FBS, 20 ng/ml interleukin-3 (StemCell), and 3 ng/ml GM-CSF (StemCell) for 5 days. Each colony formed at the end stage of differentiation was picked up by pipette tips, resuspended in 500 µl IMDM, and transferred into a 4-well chamber slide for cell adhesion at 37oC for 3 hours before Giemsa staining (Sigma) and histological examination. Phycoerythrin (PE)-conjugated anti-CD45 (Pharmingen; San Diego, CA; http://www.bdbiosciences/pharmingen.com) was used to examine differentiated cells for expression of CD45 under a fluorescence microscope.
Activation and Culture of Human Metaphase II (MII) Oocytes
Twenty-five human MII oocytes were collected from two female ovum donors who underwent downregulation with leuprolide acetate (Lupron; TAP Pharmaceuticals; Deerfield, IL; http://www.tap.com) and then began controlled ovarian hyperstimulation by receiving follicle stimulating hormone (Gonal-F; Serono; Rockland, MA; http://www.serono.com) treatment at a dosage of 300 IU/day to induce an appropriate multifollicular response. When ultrasonographic criteria for follicular maturity were met, a single 10,000 IU dose of hCG (Profasi; Serono) was administered, and transvaginal follicular aspiration was performed approximately 36 hours after hCG administration. Cumuli were removed from the oocytes by brief 80 IU/ml hyaluronidase exposure, and mature MII oocytes were subjected to three different activation procedures: A) 5 minutes exposure to 5 µM calcium ionophore (A23187, Sigma) in HEPES-buffered human tubal fluid supplemented with 10% human serum albumin (HEPES-HTF-HSA; InVitroCare; San Diego, CA; http://www.invitrocare.com) at 33°C followed by 3 hours incubation with 1 mM 6-DMAP (Sigma) in IVC-TWO medium (InVitroCare) at 37°C; B) 5 minutes exposure to 5 µM calcium ionophore in HEPES-HTF-HSA followed by 2 hours incubation with 10 µg/ml puromycin (Sigma) in IVC-TWO at 37°C, and C) sham intracytoplasmic sperm injection (ICSI) followed by 15 minutes exposure to 50 µM calcium ionophore. The activated oocytes were incubated in IVC-ONE medium for 3 days before transferring to IVC-THREE medium for an additional 2 days culture. On day 6, assisted hatching was performed using a microscope-mounted micromanipulator to expel acidified tyrodes solution (Medi-Cult, Jyllinge, Denmark; http://www.medicult.com) on the zona pelucida. The blastocysts were then allowed to hatch in IVC-THREE medium and were cultured on mitomycin-C-treated murine embryonal fibroblast (ATCC) feeder in mouse stem cell medium supplemented with 1,000 U/ml human recombinant LIF.
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RESULTS |
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depicts the derivation of a homozygous stem cell line and its advantage in immunohistocompatibility. Aiming at exploring the potential application of such homozygous stem cells and their derivatives in allogenic transplantation, mouse homozygous stem cell lines were established and cells were assessed for their stemness and pluripotency. Three cell lines were established from calcium ionophore- and 6-DMAP-treated MII oocytes of two C57BL/6J (line LC57) and three C57BL/6J x DBA/2J F1 (lines L5 and L10) female mice superovulated with the administration of PMS and hCG. All three lines formed colonies when cultured on mitotically inhibited murine embryonic feeder cell layers in medium supplemented with LIF, and colonies repropagated when trypsin digested into smaller aggregations. They were also able to form EBs when feeder and LIF were removed from the culture. All lines survived in culture for at least 15 passages, and they all retained a normal karyotype when cells from passage 10 were examined (Fig. 2A). Upon genotype analysis with markers for D2mit42 and H-2 markers (H-2-A- and D17mit232), homozygosity in the established cell line was revealed: L5 (derived from C57BL/6J x DBA2 F1 mice) was homozygous for a 150-bp allele of D2mit42 and a D haplotype for both H-2-A- and D17mit232. L10 (derived from C57BL/6J x DBA2 F1 mice) was homozygous for a 136-bp D2mit42 allele and a B haplotype for both H-2-A- and D17mit232. LC57 displayed homozygosity on D2mit42, H-2-A-, and D17mit232 identical to the C57BL/6J mice (Table 1). Expressions of SSEAs, Oct4, and telomerase were also examined. All three lines displayed positive expression of alkaline phosphatase, telomerase, and Oct4 (Fig. 2B, 2C, and 2E, respectively). This expression pattern of stem cell markers is comparable with mouse embryonic stem cells [8].
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depicts a representative histological section and the tissue components derived from line LC57.
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). The result indicates that the established cell lines could respond to endoderm lineage differentiation induction.
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). The neuronal cell morphology and the expression of tyrosine hydroxylase (Fig. 4B-4C) could also be observed after further culture in N2 medium on poly-L-ornithine, a fibronectin-precoated 24-well plate, and the removal of bFGF.
For examining mesodermal differentiation potential, an established methylcellulose-based hematopoietic cell differentiation environment was used [13–15]. In this differentiation environment, EBs were formed in the presence of monothioglycerol, cultured in M3434 medium, and subsequently cultured in GM-CSF-supplemented IMDM. CD45+ cells could be seen within the colonies when immunocytochemical analysis with a PE-conjugated primary antibody was performed (data not shown). Histological examination of single cells from the differentiated colonies revealed cells bearing the morphology of lymphocytes, monocytes, and erythrocytes, respectively (Fig. 4C). Further culture of any of the differentiated lymphoid cells was not attempted in this study.
Activation and Culture of Human MII Oocytes
Although reagents such as calcium ionophore, 6-DMAP, and puromycin have been reported to induce human oocytes to 1-pronuclear (1PN) stage [16,17], whether such activated human oocytes have the capacity to form blastocysts with substantial inner cell masses has not been well studied. To investigate the feasibility of using such reagents to activate human MII oocytes to produce viable inner cell masses for stem cell derivation, 25 human MII oocytes collected from two female ovum donors were subjected to three different activation methods: A) 5 minutes exposure to 5 µM calcium ionophore followed by 3 hours incubation with 1 mM 6-DMAP (14 oocytes); B) 5 minutes exposure to 5 µM calcium ionophore followed by 2 hours incubation with 10 µg/ml puromycin (5 oocytes), and C) sham ICSI followed by 15 minutes exposure to 50 µM calcium ionophore (6 oocytes). Among the 14 oocytes treated with calcium ionophore and 6-DMAP, we observed the development of four late blastocysts with substantial inner cell masses. So far, further culturing of the inner cell masses produced by this activation method has not been successful. In the five oocytes treated with calcium ionophore and puromycin, only one oocyte could progress to the early morulae stage. Among the six oocytes receiving sham ICSI and calcium ionophore, one developed to a late blastocyst and, after assisted hatching and further culture, gave rise to proliferating cells that survived more than two passages. Figure 5 shows the process of deriving proliferating cells from human MII oocytes.
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DISCUSSION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Mouse uniparental stem cells have been derived from gametes of only one of the two sexes (i.e., sperm or oocytes), and their derivatives have provided a unique resource for understanding developmental biology, such as imprinting, but their pluripotentiality has not been well characterized. In this study, we not only demonstrated that a metaphase II oocyte could be used to generate an MHC homozygous stem cell, but also showed that the derived stem cells were pluripotent. These cells retained a euploid karyotype, expressed stem cell markers SSEA-1, Oct4, alkaline phosphatase, and telomerase, and upon being subjected to teratomatous growth, the undifferentiated cells developed into various tissue types encompassing all three germ layers. They also responded to in vitro differentiation systems for endodermal, neuronal, and hematopoietic lineages. Adding to the recent establishment of a parthenogenetic stem cell line in nonhuman primates and its ability to differentiate to neuronal cells in vitro [18], our demonstrations of in vitro ectoderm, endoderm, and mesoderm differentiation potential in mouse MHC homozygous stem cells promise a unique solution to overcoming immune-mediated rejection in cell-based therapies.
Derivation of pluripotent stem cells from human oocytes has been explored very little. A recent study reported that blastocysts from human oocytes activated by 4 minutes of 5 µM ionomycin followed by 3 hours of 2 mM 6-DMAP were deficient in inner cell masses [19]. Whether the lack of inner cell mass was caused by activation methods or oocyte quality was not known. In our studies, we subjected human mature oocytes to potent reagents, calcium ionophore/6-DMAP and calcium ionophore/puromycin, respectively. In addition, we performed sham ICSI to begin oocyte activation before calcium ionophore exposure. Substantial activation could be observed, but no development beyond the morulae stage could be observed when puromycin was used. High-grade blastocysts with distinctive inner cells masses could be produced on day 6 in both calcium ionophore/6-DMAP (in 4 of 14) and sham ICSI/calcium ionophore (in one of six) treatments. Upon culturing the hatched blastocysts on mitotically inactivated feeder derived from mouse embryonic fibroblasts, one inner cell mass gave rise to proliferating cells that survived beyond two passages. Although the conditions for establishing a human oocyte-derived stem cell line require further research, our result clearly reveals the feasibility of coaxing an unfertilized human oocyte into a blastocyst with an inner cell mass that has the potential to be further manipulated for stem cell production.
Using unfertilized human oocytes as a source for stem cell derivation is less controversial than using fertilized embryos; it avoids the ethical concerns surrounding human embryonic stem cell research. Without the contribution from a sperm, the oocyte has a unique advantage of homozygosity, which renders its derivatives less immunogenic and provides a broader match with different MHC phenotypes. In addition, stem cells derived from unfertilized oocytes could also be selected for homozygosity of a drug response gene, a disease gene, or a cancer gene from a female carrier and, therefore, could provide a model and business rationale for drug testing and drug discovery. For example, a collection of stem cells homozygous for different drug metabolizing gene variants could be used to prescreen a drug for its prospective toxicity and efficacy in the population. A cancer progression model can be established by differentiating stem cells homozygous for a cancer gene to the cancer tissue types, leading to the identification of cancer progression markers and, perhaps, cancer prevention drugs. Furthermore, these homozygous stem cells could be used in facilitating linkage studies and in verifying the function of a single nucleotide polymorphism.
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REFERENCES |
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