Recreating living, breathing dinosaurs from a fragment of theirJurassic-era DNA is still science fiction. Creating a live mousefrom a single stem cell is, as of now, a scientific fact.
Instead of DNA, the developmental biologists at Toronto'sMount Sinai Hospital started with a single embryonic stem cell.They recovered it from the cluster of primordial cells intowhich the fertilized egg had grown by day 3.5 after mating. Atthis early stage, each such stem cell is still totipotent P- not yetcommitted to generating a specific tissue or organ. Thechromosomes in its nucleus are still capable of flowering into afull-blown, full-grown mouse.
How the Toronto team accomplished this unprecedented featthey re-port in the current issue of the Proceedings of theNational Academy of Science (PNAS). Their paper tells the tale:"Derivation of completely cell culture-derived mice from early-passage embryonic stem cells." Its lead author, Andras Nagy ofMount Sinai's molecular and developmental biology division,told BioWorld: "We really didn't know the full potential of thiscell line. We merely suspected that it might make a mouse."
Nagy added: "This is the first and full demonstration that thiscell line has the full potentiality to make a mouse, with theplacenta provided." His mention of the placenta hints at thesteps he and his co-workers had to devise and follow in theirmake-a-mouse user's manual.
Their step-by-step procedure started with 161 embryonic stemcell aggregates and ended up with three live mice. This trio ofsurvivors, the paper states, "are more than one year old. Theydo not show any signs of premature aging or tumordevelopment. All are fertile, producing normal-size litters, andexclusively transmitted the R1 genotype to their offspring."
"R1" stands for the most productive of the four embryonicstem-cell lines the Toronto group set up. The other three, withvarious shortcomings, fell by the wayside.
To tease a single embryonic stem cell (ESC) into shaping up asa complete rodent, here are the steps Nagy and his colleaguesfollowed:
1. Mate female mice with male mice.
2. After 3.5 days, flush out the pregnant female's uterus torecover her blastocysts (hollow spheres of cells). From these,grow the ESC line, especially R1.
3. Separately, mate male and superovulating female albino-strain animals. From their progeny, form tetraploid embryos, toendow the ESC line with its missing placenta-forming gene.Because the ESCs are harvested 3.5 days post-coitum, they arenot able to develop placentas because this capability branchedoff from the blastosphere cells at 2.5 days.
4. Flush the oviducts of the now-pregnant albino females toobtain two-cell late-stage diploid embryos.
5. Fuse their blastomeres by electric pulse. By 24 hours later,most of these double embryos have developed to the four-cellstage, with twice the normal chromosome number (two fromeach parent, i.e., tetraploidy).
6. Sandwich loose clumps of ESCs between two tetraploidembryos "in aggregation wells made by pressing a darningneedle into the plastic bottom of the culture plate." In their co-culture, the diploid ESCs will overwhelm the tetraploid cells,after co-opting their placenta-forming ability.
7. Transfer these packaged embryos into the uteruses of 2.5-day pseudopregnant surrogate mothers.
8. Place the fetuses born alive up for adoption by lactatingfoster mothers.
9. Take the tail tip and a blood sample from each live pup foranalysis, to identify those born of ESC lines from those withtetraploid lineage.
This identification, essential for demonstrating the success ofthe make-a-mouse experiment, relies on two detectiontechniques, one electrical, the other visual: By electrophoresingtissue samples from blood and tail tips, the Torontans coulddistinguish two isomeric variants of a key enzyme -P glucosephosphate isomerase -P that sets the two strains of starter cellsapart.
The lower-tech, visual marker is coat color -P albino for thetetraploidal progeny, pigmented for the ESC pedigree.
What makes this system, in Nagy's words, "the basis for a newrevolution in genetics" is that the ESC line can contribute notonly to the live animal's somatic cells but to its germ-linesperm or oocytes as well." He explained: "This depends on thesex of the cell line, which in turn depends on the sex of theoriginal embryo. If a male embryo, it contributes to the sperm;if a female, to the ovum."
If a sperm from such an ESC-generated male fertilizes an egg,in the next generation, Nagy continued, "you introduce thegenome of the cell into an animal, transmitting whatever genesyou manipulated into its next-generation progeny."
If this sounds a lot like germ-line therapy, Nagy agrees: "Thewhole area has a promise that should lead to such a thing."
He foresees, for example, generating hematopoietic or myoblastcell populations, and transplanting them therapeutically totreat blood disorders or muscular dystrophy.
Because ESC culture is permanent, it can grow literally millionsof stem cells. In such a large number, site-specific mutation is apractical proposition. Such gene targeting, he points out, "cancreate a mutation which doesn't exist in the mouse, such as ahuman disease model."
This is not the same strategy as forming a transgenic mousecarrying a human gene. "We humans," Nagy said, "have a 90- to95-percent overlap between our genes and the mouse genome."This makes it possible to engi-neer, for example, a cystic fibrosisrodent model to further future therapy.
This success story is a success only up to a point, and that pointis the way the R1 embryonic stem cell line wimps out after 14passages in culture. For reason or reasons unknown, itstotipotency declines after that milestone, and no viableprogeny survive if derived from more than 14 passages. This,as the PNAS paper concedes, makes the process, "inefficient." Itneeds work, and the Toronto team is on its case, optimistic thatit will eventually contrive "a feasible approach for routinelyachieving germ-line transmission from genetically manipulatedES cells."
It was 12 years ago that Martin Evans and Matthew Kaufmanin Britain and Gail Martin at the University of California, SanFrancisco, simultaneously showed it was possible to establishpermanent cell culture from very early mouse embryos..Thomas Rosenquist, a post-doc in Gail Martin's lab at UC/SF,told BioWorld anent the Nagy report in PNAS: "If they can findculture conditions that would allow you to reliably culture EScells and maintain their totipotency, that would be a bigbreakthrough." Acknowledging Nagy's "first" in making a wholemouse from a single cell, Rosenquist added: "Becausetechniques are getting simpler and simpler by other methods,it probably isn't going to be a general method for generatingmice."
-- David N. Leff Science Editor
(c) 1997 American Health Consultants. All rights reserved.