Stem Cells... An Emerging Portrait

PRIMORDIAL BEGINNINGS?

Pluripotent cells are uncommon and ephemeral in embryos, nestled among blastomeres, ICM cells, primitive ectoderm, and primordial germ cells (PGCs). They are essential to maintaining the high cell-division rates that build a new body. The culturing process isolates, captures, and sustains these abilities. So to understand ESCs, a good start is to trace embryogenesis to see which cells they most closely resemble. And so far, it looks like hESCs are not most like ICM cells.

An early clue that hESCs might most closely resemble PGCs, or their precursors, came from Leroy Stevens, who worked on teratoma-prone mice at the Jackson Laboratory from the 1950s through the 1980s.² Stevens meticulously dissected mouse embryos in search of the cells that led to formation of teratomas – hideous multitissue growths, usually in a testis. Jackson's critical leap was recognizing the close resemblance of the responsible pre-PGCs to cells of the early embryo. In a masterful series of experiments, he implanted early embryo cells, including those of the ICM, into adult testes. As he anticipated, teratomas formed. Stevens named these ICM cells "pluripotent embryonic stem cells," hence coining the now-familiar term, but it was quickly forgotten because the cells also gave rise to cancer cells in some teratomas. The besmirched cells became known as embryonal carcinoma, or EC, cells. A teratoma without EC cells is benign; with EC cells, it is a teratocarcinoma. EC cells proved difficult to nurture in blastocysts to see if they could support development because of chromosomal instability. Nevertheless, they could become part of normal chimeras on rare occasion.

In 1981, Gail R. Martin at UCSF, and Martin Evans and Matthew Kaufman at Cambridge University, isolated EC cells that had stable chromosomes and had apparently escaped the cancer taint.³⁴ These were mouse ESCs, which became the foundation of knockout technology and led the way to the derivation of hESCs.⁵⁶ Amander T. Clark, a research geneticist in Reijo Pera's group, distinguishes between these two types of pluripotent cells. "EC cells have chromosome abnormalities, and this is probably why you run into trouble when injecting them into blastocysts. ESCs do not have the chromosomal abnormalities, but have the same potential as EC cells."

This past year, Reijo Pera, Clark and their colleagues compared transcriptional profiles of three hESC lines to human ICM cells, painting a molecular portrait that supports the germ-cell origin hypothesis.⁷ "Markers of germ cells are expressed in hESCs and not in ICM cells. The implications are that hESCs are likely derived by selection of a retained small population from the ICM, or by ICM cells developing beyond their original stage," says Reijo Pera.

The researchers discovered, for example, that undifferentiated hESCs produce DAZL, which is a germ-cell specific marker that acts premeiotically. They also found that hESCs implanted in mouse peritoneal cavities, where they give rise to embryoid bodies, display more differentiated germ-cell markers, such as VASA. Moreover, while hESCs and ICM cells express three markers in common (NANOS1, STELLAR, and OCT4), hESCs alone sport certain stage-specific embryonic antigens. And so assuming that hESCs are most like ICM cells because that is their immediate source may be more guilt-by-association than reality.

Even if hESCs most closely resemble PGCs, or pre-PGCs, they are not the same as these in vivo cells. ESCs self-renew longer than PGCs, and, when placed in chimeras, support normal development. Culture conditions probably help to mold stemness.⁸ Clarifying the relationship between ESCs and PGCs, however, requires observing development of human embryos into what some consider unethical territory – beyond 14 days, when formation of the primitive streak heralds the first inklings of a nervous system.

EMERGING PORTRAIT

Beyond the two most important defining characteristics-self-renewal and pluripotency-stem cells also have multiple nucleoli, a low cytoplasm-to-nucleus ratio, and lipid bodies in the cytoplasm. In a general sense, hESCs express 20% to 30% of the genome, compared to 10% to 20% for most somatic cells. The most highly expressed genes in hESCs encode proteins involved in cell-cycle control, apoptosis, DNA repair, development, and stress responses – a rather generic laundry list. But by using DNA microarrays, serial analysis of gene expression (SAGE), and the more powerful massively parallel signature sequencing (MPSS), researchers are homing in on specific genes whose products impart a state of stemness While DNA microarrays can only glimpse the sequences chosen for probing, SAGE and MPSS pull out thousands to millions of transcripts from a cell, and can spot splice variants.

In one recent study, Ariff Bongso, research professor in the department of obstetrics and gynecology at the National University of Singapore, and colleagues used SAGE to compare two hESC lines and a mouse ESC line. They identified 21 candidate hESC marker genes, including 10 novel ones. The two human lines were very alike in gene expression, despite their derivation from individuals of different sex and ethnicity.⁹

Whatever hES cells are, or aren't, might become clearer in mid-August, when researchers from 17 labs representing 10 countries present profiles of 75 hESC lines at the first meeting of the International Stem Cell Initiative at the Jackson Laboratory in Bar Harbor, Maine. Of special interest, says coordinator Peter Andrews, professor of biomedical science at the University of Sheffield, is the diversity of hESCs compared to the few closely related mouse strains.

Until then, actual description of hESCs remains murky. Says Hayek, "Human ESCs are real. What is artificial is their environment. And that is what will prove an extremely difficult nut to crack."