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Personal pathways to embryonic stem cells
Work with numerous colleagues on embryo stem cellsin vitrobegan in our laboratories in 1963, using cleaving embryos and blastocysts of the rabbit. Growth of disaggregated blastomeres from cleavage-stage embryos was weak. Trophoblast outgrowths from whole-embryo cultures provided a platform supporting inner cells that differentiated into blood islands, muscle, nerve cells, phagocytic cells, connective tissue and undefined elements. Stem cell lines derived from whole or disaggregated cells of rabbit inner cell mass and embryonic disc proved to be long-lasting (immortal), growing over months and years, stable enzymically, karyotypically and morphologically, and fully capable of resuming growth after cryopreservation. To measure the embryological potency of embryo stem cells, disaggregated or entire inner cell masses were injected into the blastocoelic cavity of mouse blastocysts. They contributed to the formation of chimaeras, and partially colonized most or all recipient tissues except trophectoderm. Gene markers for coat colour enabled their instant detection in recipient fetuses, newborns and adults. Cell lineages, the multipotency of single cells, and transgenesis all descended from this approach. Mouse and rat stem cells were grown from disaggregated blastocysts culturedin vitroor from tissues extracted from early post-implantation mouse embryos. They fully recolonized bone marrow in lethally irradiated adult mouse recipients, and in mice carrying inherited anaemias, migrating via liver to bone marrow and spleen. Some may have been hepatocyte or splenocyte precursors. Non-irradiated recipients were weakly colonized by embryo stem cells overcoming histocompatibility barriers. No signs of inflammation or cancer were noted. First studies on human embryo stem cells began, but were ended by an ethical decision preventing supplies of human blastocysts for research.
Keywords: colonization of recipients, embryo stem cells, grafting, history, human, mammals.
Section 1. Introduction
Embryo stem cells are not a recent matter. They emerged coincidental with the beginnings of IVF, and as new understandings of the growth of preimplantation embryos, especially mouse, were being initiated. This paper briefly describes the emerging knowledge on mammalian embryos in the mid-twentieth century, when ideas first arose of growing stem cells and using them to repair sick or deficient tissues in recipients. Much of this paper involves my own involvement in this formative period, already described in brief elsewhere (Edwards, 2001).
Five successive studies will be outlined. An initial section describes growing knowledge on preimplantation biology over these years. A description of the novel opening studies on rabbit embryo stem cells follows, revealing their capacities for differentiationin vitroand into the first embryo stem cell lines. A new approach, developed to measure embryological potentials of single or groups of cells from inner cells mass, involved their injection into the blastocoelic cavity of mouse blastocysts. Final sections include the initial use of embryo stem cells to recolonize bone marrow, liver and spleen in sick adult mice and the beginnings of practical and ethical work on human embryo stem cells.
Section 2. First attempts at making embryo stem cells
Section 2.1. Preimplantation embryology becomes a scientific study
Studies on preimplantation embryology of mammals began to expand in the 1950s, coincident with sharp advances in amphibian embryology. Heape in 1890 had obtained offspring after transferring rabbit blastocysts into recipient does, based on work by earlier pioneers. Rabbit embryos were cultured through early cleavages, and on to morulae and blastocysts (Lewis and Gregory, 1933; Lewis and Wright, 1935). Rabbit oocytes released from their follicles into culture media matured spontaneously to metaphase 2 (Pincus and Enzmann, 1935). The destruction of one blastomere in 2‐cell embryos revealed the other to be capable of sustaining fetuses to term (Nicholas and Hall, 1940; Seidel, 1952). Beatty and Fischberg (1951), working in the Institute of Animal Genetics, Edinburgh, warmed fertilized mouse eggs within the oviduct in order to destroy the second meiotic spindle. The second polar body was thereby retained in the egg, to form triploid embryos. Haploids, triploids and occasional tetraploids arose among aneuploid forms (Beatty, 1957). I became Alan Beatty's PhD student, and modified his treatments to produce androgenetic and gynogenetic haploid embryos, triploids, tetraploids and various heteroploids (Edwards, 1957).
Stimulated by this work, my interest in mammalian embryology was established, and the Institute was an excellent place for new studies on mice highly relevant to later work on stem cells. Radiolabelled precursors of DNA, RNA and proteins, combined with autoradiography, measured their cytochemical distribution and turnover (Edwards and Sirlin, 1956). Gradients in RNA and proteins, typical of amphibian oocytes, could not be identified in mouse oocytes in cleaving embryos and blastocysts (Edwards and Sirlin, 1956). Labelling DNA in the final synthetic period in mouse spermatocytes marked them precisely, enabling their incredibly consistent progression through meiosis and spermiogenesis to be estimated, and providing insights into strictly timed developmental processes (Sirlin and Edwards, 1955, 1958). Similar studies on oogenesis revealed all primary oocytes formed before birth (Sirlin and Edwards, 1959). Closely-timed systems were also found in the ovary when adult mice were stimulated with gonadotrophins, using methods devised by Smith and Engle (1927), Runner and Palm (1953) and others. Multifolliculation, oestrus and ovulation followed in a precise succession (Fowler and Edwards, 1957). A strict timetable also characterized oocytes maturing through germinal vesicle breakdown, meiosis‐1 to extrusion of the first polar body, and to ovulation beginning at 11.5 h after an ovulatory injection of human chorionic gonadotrophin (HCG). Fertilization, cleavage, blastulation and implantation were also closely timed (Edwards and Gates, 1959). I was now interested in applying this work to human studies, even though failing to repeat the work of Pincus and Saunders (1939) on maturing human oocytesin vitroin 12 h (Edwards, 1962, 1965). Chang (1955) matured rabbit oocytesin vitro, and transferred them to oviducts of mated females, to show how they could not implant unless initially exposed to HCG.
Ideas on differentiation in mammalian embryos expanded as Tarkowski (1959, 1961), Mintz (1964) and others disaggregated or fused 2‐, 4‐ and 8‐ and 16‐cell mouse embryos to produce chimaeric offspring. Their findings, and others, showed mammalian blastomeres were highly multipotent, and inner cell mass cells retained considerable potency as they were committed in succession to specific embryological fates. These ideas led me to begin work on individual blastomeres by disaggregating cleaving mouse and rabbit eggs for in-vitro culture. They divided as separate cells, not for long, yet just enough to provide hints of new approaches to establishing stem cell lines (Edwards, 1964). Rabbit blastomeres and embryos proved easier to culture than mouse cells.
Section 3. Rabbit embryo stem cells
These initial studies led to detailed collaborative studies on stem cells with Robin Cole and John Paul. In his superb cell culture facility, John maintained many cell strains in continuous culture, utilized modern biochemical methods, and practised strict tissue culture including the use of CO2incubators and novel techniques such as holding microdrops beneath paraffin overlays. We decided to concentrate on blastocyst cells, especially inner cell mass, as a source of differentiating cells and stem cells.
Section 3.1. Cell outgrowths from cleavage embryos and blastocysts
Disaggregates of single or grouped preimplantation rabbit embryos were cultured in droplets of NCTC109, 199, F10, Eagle's, and Waymouth's media held under paraffin oil overlays. Supplements included sera from fetal calves, human or rabbit serum and occasionally irradiated HeLa feeder cells (Coleet al., 1965, 1966). Some glass or plastic culture vessels were coated with collagen (Ehrmann and Gey, 1956), and a mesodermalizing inducer was added to some colonies (Tiedemann and Tiedemann, 1959).
Disaggregates from several 4‐ to 8‐cell zona-free rabbit embryos grouped in culture droplets occasionally produced a migratory cell type. Better growth was obtained with 8‐ to 16-cell stages. Still better growth with 32‐cell stages and morulae produced more examples of this cell, which frequently attached to and formed a pavement on culture dishes ( Table 1 ). A few rounded, perhaps embryonic-like cells, did not persist (Coleet al., 1965, 1966). When embryo structure was retained in culture, some 4‐cell colonies produced small or misshapen blastocysts after 5 days, their trophoblast attaching to culture dishes to form epithelial cell outgrowths.
|Embryo stage||No. of cultures||No. showing attachment|
Intact zona-free rabbit blastocysts were cultured, sometimes with a collagen substrate or mesodermalizing inducer. Their structure was distorted as they attached to the culture dish. Most shrank as their blastocoelic cavities collapsed, expanding and collapsing again (Coleet al.1965, 1966). Cells migrating rapidly from the attachment site pulled remaining trophoblast to the dish surface overnight, transforming embryos to thin cell sheets with a central bundle of cells ( Table 2 ). The migrating trophoblast pavement even pushed feeder cell layers back, and may have been phagocytic (Maximov 1925). Smaller-sized blastocysts produced trophoblast only; they may have been trophoblastic vesicles when placed in culture. Twelve trophoblastic cultures were diploid and two were tetraploid. Smaller round cells, presumably inner cell mass, migrated over trophectodermal pavement from the persisting cell mass in the centre. These masses were later called embryoid bodies. Central cells often formed a second cell layer often highly organized and covering trophoblast (Coleet al., 1966). Structures and vesicles formed and regressed. Most colonies regressed after a static period.
|A. Outgrowths of embryo stem cells from whole rabbit blastocyst cultures|
|Difficult to establish from 1‐cell and cleavage stages|
|Blastocysts adhere to culture vessels|
|Inner cells migrate over trophectoderm forming piles of cells and outgrowths|
|Tissues resembling muscle, connective tissue, blood islands, neurons, macrophages|
|Possible role of collagen and ‘mesodermal inductors’|
|B. Establishing rabbit embryonic stem cell lines in vitro|
|Embryonic discs excised from blastocysts|
|Some cultured intact between slightly compressed glass surfaces|
|Others disaggregated into separate cells|
|Encouraged to attach by providing collagen-coated surfaces|
|Piles of cells and outgrowths|
|Twenty stem cell cultures possible from single embryonic disc|
|Forty sub-cultures initially, some passing through estimated 200 generations|
|C. Properties of long-lasting rabbit embryo stem cell lines in vitro|
|Name||Origin||Time to establish||Characteristics a||Enzymes||Persistence|
|RB1||Day 6, outgrowths of embryonic disc||6 weeks||Fibroblastic spindle-shaped||High arginase||>11 months or 200 generations|
|RB2||Day 6, outgrowths of embryonic disc||6 weeks||Fibroblastic, diffuse|
|RB3/3||Day 6||Epithelial High||Alkaline||>9.5 months|
|RB3/4||Day 6||Fibroblastic, as RB1||phosphatase|
a RB1 and RB3/3 remained largely diploid.
NB Cell lines cyopreserved and thawed without loss of characteristics.
Fixed and stained preparations of blastocyst colonies revealed how various tissues had differentiatedin vitro(Coleet al., 1965, 1966; Edwards, 1980). Blood islands identified after 12 days post-attachment ( Figure 1 ; Table 2 ) were related to structures typical of yolk sac. Overlying massive sheets of muscle-like cells were identified, but made only minor if any contractions. Individual cells resembled neurons, macrophages apparently with ingested material, connective and other tissues stained with Alcian blue or Mallory's triple stain. Vesicles and solid structures could not be classified. Embryo cells clearly had immense potentialin vitro, the inducer possibly stressing growth towards mesoderm. Clear thoughts of using stem cells in clinical medicine were emerging in mid-1963 ( Figure 2 ).
Section 3.2. Rabbit stem cell lines from isolated or disaggregated inner cell mass
In an alternative approach, inner cell masses were isolated or disaggregated in attempts to establish embryo cell lines. Zona-free rabbit blastocysts 6 days post-conception were gently opened with glass needles, avoiding damage to their constituent cells. Cuts around inner cell mass excluded most trophectoderm cells (Coleet al., 1965, 1966). The isolated inner cell masses were placed in droplets of medium on collagen-coated cover slips beneath liquid paraffin, their inner surfaces adjacent to collagen. Attachment was encouraged by removing culture medium so that liquid paraffin exerted pressure on the inner cell mass. Most of these masses attached within 10–15 h, when droplet volumes were increased.
Sheets of cells now formed, usually surrounded by a few trophoblast cells. If trophoblast cells became too numerous, inner masses of cells were cut out and transferred to fresh medium. This dense central mass of cells may have grown over a few supporting trophoblast cells. Cell monolayers migrated from the central mass within 4–5 days post-attachment, and formed epithelial and fibroblastic cells. If the central cell mass degenerated, surrounding cells formed a monolayer over the available collagen surface, then attached directly to collagen-free surface as fibroblasts. Some outgrowths were trypsinized, the disaggregated cells being transferred to new cultures. Two primary fibroblast-like cultures were isolated and maintained on glass, one at 6 weeks after initial explants migrated from cultures of 6‐day blastocysts ( Table 2 ) (Coleet al., 1965, 1966).
Embryonic discs of day 6 blastocysts were then disaggregated using trypsin and cultured singly on collagen-coated coverslips in a culture dish, with a gas phase of 5% CO2in air. Cells attached and spread over collagen in 24 h, none attaching directly to glass or plastic. Small colonies of epithelial and fibroblastic cells were identified after 5–7 days, and up to twenty epithelial and fibroblastic cell lines were established from a single embryonic disc ( Table 2 ). Many colonies died. No colonies were established from embryos of younger ages (Coleet al., 1965, 1966).
The growth characteristics of established cell lines were most unusual. They transmitted to glass 6 weeks after explantation, and many lines grew strongly, seemingly held at a specific differentiation stage. The fibroblastic line RB/1, and the epithelioid line RB3/3, retained their morphology and contained large nuclei and many nucleoli ( Table 2 ). Most of the cell lines examined were diploid, two being tetraploid. Cell lines typed for high levels of alkaline phosphatase or arginase activity retained their enzymic character over many generations. Two lines became immortal, cleaving endlessly over months and even years. Cryopreserved and thawed samples immediately resumed division when thawed, some after several years in cryostore (Coleet al., 1965, 1966; J. Paul, personal communication 1975).
Less success was gained with mouse embryo cultures in our and in other laboratories. Two-cell embryos grew through cleavage to blastocysts, and to hatching. All 50 expanding blastocysts cultured in one microdrop had hatched by next morning (Cole and Paul, 1965). With extended culture, inner cell mass became malformed, jeopardizing the establishment of stem cell lines as trophoblastic vesicles formed as found earlier (Tarkowski, 1959; reviewed by Sherman, 1975). Studies on the biochemistry of different cell lines and trophectoderm derivatives were also jeopardized (Sherman, 1972, 1975), although at this time, mushroom-shaped clumps of embryonic cells formedin vitroand some persisted over long periods. Stevens (1967) noted that intraperitoneal injections of EC cells (embryo teratocarcinoma cells) to syngeneic hosts produced ‘embryoid bodies’ containing multipotent cells.
Collagen substrates used by Coleet al.(1965, 1966) must have been significant in establishing rabbit stem cell lines. Today, substrates are selected to modify gene expression, and commit stem cells to various pathways. One example utilized cells derived from freshly explanted bovine mammary epithelial cells (Delabarreet al., 1997). Matrigel basement matrix from mouse EHS tumours favoured the formation of alveolar structures but not tight junctions. Fibronectin encouraged a typical pavement architecture and transepithelial electrical resistance in tight junctions. A laminin surface invoked the diappearance of αS1‐casein after 5 days and a stable transepithelial resistance after 15 days. With collagen‐1, an epithelial pavement formed within 2 days, casein was secreted from cell apices over 2 weeks, and epithelial resistance remained stable (Delabarreet al., 1997). Rabbit trophectoderm, basically an epithelium, had established similar pavements on collagen and plastic surfaces (Coleet al., 1965, 1966).
Data gained in these opening studies in the early 1960s formed the basis of later work with stem cells ( Figure 2 ). Methods were needed immediately to assess the developmental properties of single embryonic stem cells. Mice offered wider and simpler opportunities than rabbits, and their embryos formed fusion chimaeras with donor cells, described above, and helped in tracing developmental fates of single cells. Any clinical application of embryo stem cells would have to await the successful maturation and fertilization of human oocytesin vitro(Edwards, 1962). By a remarkable coincidence, the first human oocyte matured to metaphase‐2, and extruded its first polar body, during the final days of the Glasgow period. I moved to Cambridge with these stimulating thoughts in mind ( Figure 3 ).
Section 4. Mouse stem cells: injections into blastocoelic cavity of recipient blastocysts
A small mouse house was available in Cambridge. Mice were inexpensive, well typed genetically, and produced many embryos in response to ovarian stimulation with gonadotrophins (Runner and Palm, 1953; Fowler and Edwards, 1957). They fulfilled the need for inbred lines and strains carrying marker genes such as coat colour, chromosomal and biochemical genetic markers to identify descendants of donor and recipient cells. Two possible approaches promised to measure developmental potentials of single stem cells. One involved fusion chimaeras, already achieved by fusing isolated cells with cleaving mouse embryos (Tarkowski, 1961). The untested second approach was to make injection chimaeras, by injecting one or more embryo stem cells into the blastocoelic cavity, promising a more direct approach for assessing single stem cells ( Figure 3 ). It even seemed possible to classify recipient embryos by studying one or a few cells excised from trophectoderm (Edwards, 1962). These studies were ideal for a PhD project, and research by postgraduate students was essential in realizing this and other ideals in Cambridge. I was indeed fortunate to work with Richard Gardner, Martin Johnson, Roger Gosden, Matt Kaufman, Azim Surani, Alan Handyside, Peter Hollands and others on various topics in embryology and stem cells.
Section 4.1. Micromanipulating embryo stem cells into blastocysts to form chimaeras
Richard Gardner chose to study embryo cells by making injection chimaeras for his PhD. Initial experience with micromanipulation was gained by operating on larger rabbit blastocysts. The intention was to excise a few rabbit trophectoderm cells and type them for sex chromatin, which marked female embryos. This study was successful, and introduced the preimplantation genetic diagnosis of inherited disease in humans and animals (Edwards and Gardner, 1967; Gardner and Edwards, 1968). An absence of sex chromatin in female human blastocysts thwarted similar human work 5 years later.
Gardner's methods for introducing single or groups of cells into the mouse blastocoelic cavity are familiar world-wide today. Gene markers, micromanipulators, specifically designed pipettes, and good quality microscopes proved robust, enabling entire inner cell mass or single cells to be injected into the blastocoelic cavity ( Figure 3 ). Operated blastocysts were transferred to recipient females to establish pregnancies (Gardner, 1968). Chimaeras were distinct in some offspring, weak in others, and most organs except trophectoderm were colonized in 14% of recipients ( Table 3 ). Germline was also colonized, revealed in back- and intercrosses between chimaeras and donor strains.
|No. cells injected a||No. of transfers||No. blastocysts||No. fetuses at mid-term||No. of chimaeras b|
|A. Fetuses examined at 12–14 days|
|B. Offspring of recipients|
a Isolated blastocyst cells from donor blastocysts.
b Number of chimaeras probably underestimated because only a few tissues examined.
Donor cells could be synchronous, asynchronous to a large extent, injected singly or in groups, taken from a different species (Gardner and Johnson, 1973), derived from teratocarcinomas (Brinster, 1973) or from parthenogenetic embryos (Suraniet al., 1977). Injection chimaeras, like fusion chimaeras (Tarkowski, 1961), were invaluable for studies on organ formation and assessing cell fates, especially with single cell injections. Fate maps were produced for different types of donor embryonic cell (Gardner and Papaioannou, 1975). Injecting single donor ICM cells into recipient blastocysts identified at least two of the 23 primitive ectoderm cells in day 5 mouse embryos to be capable of contributing to soma and germline (Gardner and Papaioannou, 1975). This work also proved that one X‐chromosome was inactivated soon after implantation in primitive ectoderm cells. Single-cell injections became even more powerful as gene insertion using homologous recombination was introduced, gene knock-out and knock-in becoming fundamental for many embryogenetic studies over the next 20 years (Smithieset al., 1985; Capecchi, 1980; Evanset al., 2001). Today, it is well known that stem cells isolated from various organs will form injection chimaeras and are highly multipotent.
Other lines of work were emerging. Pierce (1967) and Stevens (1968) continued work with teratocarcinomas. These cells produced embryoid bodies and stem cell linesin vitro(Evans, 1972), and displayed considerable potential in injection chimaeras (Brinster, 1973). Martin and Evans (1975) produced clonal stem cell lines from teratocarcinomas, and Evans and Kaufman (1981) produced multipotent embryo stem cell lines from mouse blastocysts, which displayed the same characteristics as rabbit stem cells (Coleet al., 1965). Mouse embryo stem cellsin vitroalso shared the capacity to produce blood, nerves, muscle and connective tissue (Doetschmanet al., 1985).
Throughout these years, human IVF was becoming a clinical reality as eggs were fertilized and cleavedin vitro. Some grew to day 5 and day 9 human blastocystsin vitro, their splendid inner cell masses and embryonic discs packed with stem cells (Edwardset al., 1969; Steptoeet al., 1971; Edwards and Surani, 1978). Even though the birth of the first IVF baby was delayed until 1978, it became a signal indicating the imminence of alleviating infertility, introducing preimplantation genetic diagnosis and developing human stem cells for therapeutic purposes. Not surprisingly, this period was near-euphoric for me as these clinical possibilities began to emerge.
Section 5. Mouse embryo stem cells: colonization of deficient organs
Animal studies were initially essential to assess the potential therapeutic value of human embryo stem cells. This project, completed in the mid-1980s, proved decisive in completing the whole scope of stem cell therapy.
Fetal tissues, especially haemopoietic cells, have long been used to colonize deficient organs in sick adults. Liver haemopoietic cells from fetuses colonized syngeneic hosts, provided persisting liver grafts, and reversed inherited anaemia in recipients (Uphoff, 1958). Injected into placental vessels of 11 to 12‐day-old fetal recipients, fetal liver cells from 13‐day mouse fetuses repaired their macrocytic anaemia (Fleischman and Mintz, 1979). Classically, the effectiveness of haemopoietic stem cells was measured by the formation of single-cell colonies in spleen (spleen colony-forming units or CFUs) (Till and McCulloch, 1961). Mesenchyme was the source of CFUs, capable of differentiating into erythrocytes, granulocytes and platelets (Harrisonet al., 1988). This was the optimal animal model to assess the therapeutic value of embryo stem cells in recolonizing bone marrow in lethally irradiated adult recipients, another challenging PhD project. Peter Hollands elected to carry it out in mice, using stem cells derived from blastocysts or early post-implantation embryos.
Section 6. Therapeutic value of stem cells from post-implantation mouse embryos
Section 6.1. Use of stem cells from newly implanted embryos to repair bone marrow in irradiated adult recipients
Initially, the developmental potential of stem cells extracted from early post-implantation embryos was measured by assessing their potential to recolonize bone marrow in lethally X‐irradiated adult mouse recipients (Hollands, 1987). Donor mouse strains included CBA, MF1, C57Bl/10 and CBA mice carrying the chromosome marker T6 (CBA-T6T6). Recipient strains exposed to lethal doses of X‐irradiation before grafting included Balb/c and strain 129 mice. Respective genetic markers included haemoglobin variants as erythrocytic markers, and variants of glucose phosphate isomerase (GPI) for lymphoid cells. Some donor and recipient strains were characterized by differing HLA types (Table 4 and Table 5).
|Series||Strain combinations||No. surviving Embryo cells||>350 days Adult bone marrow|
|136/172 (80%)||22/102 (21%)|
Eighty percent of Balb/c recipients of embryo cells had donor Hb and GPI markers.
Survivors in Series 1–3 lived for 600±30 days, and Series 4–10 were alive and well at 450 days.
In mice, erythropoiesis was considered as initiating in yolk sac from 7 days post-fertilization, continuing in liver and then in bone marrow (Weissmanet al., 1978; Burgess and Nicola, 1983). Preliminary studies identified haemopoietic stem cells in mouse embryos at day 6, slightly earlier than expected (Hollands, 1988a). Hence, entire implanted embryos at days 6–7, dissected from the uterus, were disaggregated and injected into tail veins of recipients. Each recipient received cells equivalent to three donor embryos within three hours of preparation. A total of 4.5 × 104donor cells were injected, equivalent to 104 haemopoietic cells assuming they formed 20% of the total ( Table 5 ). Subsequently, 90 µl blood was collected from recipient tail veins a few days after grafting or at weekly and longer intervals, and assessed for markers of lymphocytes and erythrocytes using marker haemoglobin and GPI.
Overall results of several trials, utilizing densitometry for haemoglobin and GPI, revealed a very rapid colonization by donor cells of recipients' liver and bone marrow (Table 5 and Table 6) (Hollands, 1987). Many recipients expressed donor haemoglobin within 2–3 days, some within 24 h. Heavily colonized recipients reached 20% colonization within days. Recipients' haemoglobin survived for 30 days when donor cells became dominant. Donor GPI appeared slightly later, from 3–4 days post-graft, reaching values of 20% in recipients by 3–4 days (Hollands, 1987). Donor cells synthesized adult haemoglobin, not fetal haemoglobin, perhaps stimulated by their new adult environment. Normally, cells synthesizing adult haemoglobin arise at day 8 of gestation in mice.
|A. Erythrocytic colonization in one series (marked by haemoglobin)|
|Days post injection||Colonization of recipients (%)|
|B. Lymphocytic colonization in one series (marked by GPI)|
|Days post injection||Colonization of recipients (%)|
|C. Detailed analyses showing the first appearances of donor haemoglobin and GPI in all series|
|Time (days) of donor GPI appearance||Time (days) of donor haemoglobin appearance|
Adult irradiated Balb/c mice were then grafted via tail veins with cells from CBA-T6T6 donor embryos. Mitoses carrying this marker revealed an initial and massive colonization of recipients' liver within 24–48 h ( Table 7 ). Donor cells persisted in liver until day 8 post-graft. They could have included hepatic precursors that remained there, and haemopoietic cells that migrated through liver to bone marrow, as in fetuses. Marker cells entered bone marrow on day 4, increasing steadily until they comprised 50–100% of mitoses in recipients (Hollands, 1987). This route clearly followed fetal pathways of haematopoietic cells. Approximately 10% of all mitoses were donor T6 cells in recipients' spleens at 64 days post-graft.
|A. Liver colonization|
|Days post-injection||Colonization of recipients (%)|
|B. Bone marrow colonization|
|Days post-injection||Colonization of recipients (%)|
Four-fifths of recipients survived to old ages, all expressing donor markers (Table 5 and Table 8). At least 8 × 105nucleated donor cells, equivalent to one embryo, were needed to sustain a successful graft. Histocompatibility differences were breached without obvious effects (Hollands, 1997). Controls died within 14 days after irradiation, and did not express donor markers. Tissues from embryos aged <6 days did not colonize, e.g. day 5 cells from MF1 donors injected into strain 129 recipients. These irradiated recipients died at 12–14 days, as with non-injected irradiated controls. Standard donor bone marrow preparations also failed to colonize any recipient, as did injections of decidual cells.
|A. Numbers of injected cells, expression of donor markers and duration of survival|
|Nucleated cells injected (×104)||Donor cells in culture (days)||No. recipients with donor markers||Recipient survival (days)|
|4.8–6.5||3||4/5||4/5||14, 17a, 315, 322, 339|
|6.7–7.1||3||3/5||3/5||13, 14, 301, 323, 341|
|7.8–9.3||4||4/5||4/5||12, 18a, 326, 335, 343|
|9.8–10.6||4||3/5||3/5||12, 13a, 14, 311, 326|
|aEach of these recipients died early despite exhibiting donor markers.|
|B. Donor haematological parameters in recipients|
|Days post-graft||Haemoglobin (g/100 ml)||Erythrocytes (×109 per ml)||Leukocytes (×106 per ml)|
Section 6.2. Use of stem cells from newly implanted embryos to repair bone marrow in non‐irradiated adult recipients
Success with grafting stem cells into irradiated mice led to attempts to engraft embryo cells into non-irradiated recipients (Hollands, 1988b). Donor cells from newly implanted embryos and assays of colonization were the same as before, utilizing strains CBA-T6T6 and C57Bl/10. Additionally, adult marrow cells were prepared from mothers of donor embryos. Recipients came from strains Balb/c and CBA. Recipients failing to accept a stem cell graft were re-injected once or more to discover if colonization could still be effected. Skin grafts were used to test for indications of rejection at 12–14 days after stem cells were injected.
Initially, 0.1–0.2 ml of mouse stem cells prepared from day 7 mouse embryos were injected into non-irradiated recipients. Among 40 injected Balb/c recipients, haemoglobin and GPI markers identified 16 (40%) of them with weak signs of colonization by 3–6 weeks after grafting ( Table 9 ) (Hollands, 1988b). Many non-colonized recipients were injected again, sometimes with a further three or more inoculations. None of them was colonized, and they survived without donor cells. Colonized recipients retained dominant levels of their own markers.
|A. Erythrocytic and lymphocytic marker expression in Balb/c recipients, related to numbers of injected cells from C57Bl/10 donors|
|No. stem cells injected (×106)||No. recipients||Donor haemoglobin in recipients (%)||Donor GPI in recipients (%)|
|Recipients with no evidence of colonization eliminated.|
|B. Migration of CBAT6T6 donor stem cells through liver to bone marrow in recipient CBA mice (Hollands, 1988b)|
|Days post-graft||No. recipients with donor mitoses in liver||No. recipients with donor mitoses in bone marrow|
The T6 marker chromosome again identified donor cells colonizing liver initially, then bone marrow where they persisted for >64 days ( Table 9 ) (Hollands, 1988b). Occasional donor mitoses at two per liver preparation appeared by day 2 post-grafting, and remained static over further days. This colonization level was lower and slower than in irradiated recipients described earlier, where stem cell mitoses were seen in liver on day 1 (Hollands, 1987). A few donor mitoses also entered recipients' spleen. Repeated failures with successive grafts might have been due to sensitization by the initial graft, other forms of sensitization, or because space was lacking for colonization in recipient liver or bone marrow. The size of the initial graft was not a cause.
Although colonization was weak, it represented the first occasion when donor cells had colonized intact recipient mice across a histoincompatibility barrier (Hollands, 1988b,c). Some recipients that accepted grafts of embryo stem cells were given skin grafts from the donor strain. All these grafts were rejected, indicating a clear difference between the rejection induced by normal grafts and its apparent absence in the presence of embryonic grafts. Allogeneic bone marrow grafts all failed to colonize the recipients (Hollands, 1988b).
Section 7. Use of cells from cultured blastocysts to repair deficient organs in irradiated recipients
Section 7.1. Use of cultured blastocyst stem cells to repair X‐irradiated adult recipients
Stem cells from blastocysts were prepared for recipients (Hollands, 1988a). Blastocysts were flushed from the uterus of C57Bl/10 mice on day 3 post-coitum and cultured in groups of 50 in 0.1 ml droplets of CMRL 1066 medium. Media additives included pyruvate, glutamine, and fetal calf serum or blood from human umbilical cords. Embryos lost their morphology in culture, and many attached to plastic. Outgrowths developed and formed embryoid bodies and blood islands in culture. Dead blastocysts were discarded.
Embryo cells were harvested after 3–4 days in culture, i.e. the equivalent of gestation days 6–7. Each drop of 50 embryos yielded 1.1–2.6 × 104 cells. These were injected into tail veins of lethally irradiated or anaemic mice recipients soon after irradiation. Controls included cell lines from blastocysts culturedin vitrofor <3 days (Hollands, 1988a), and standard ES cells prepared as described by Evans and Kaufman (1981). The same haemoglobin and lymphatic GPI markers distinguished donor and recipient combinations.
One group of irradiated adult recipient Balb/c mice received stem cell injections from cultured groups of C57Bl/10 blastocysts within a day ( Table 10 ). Many recipients displayed donor markers for haemoglobin and GPI within 3–4 days (Hollands, 1988a). Numbers injected between 4.9 and 10.5 × 104were equally effective, irrespective of their culture for 3 or 4 days. Among them, 90% with successful grafts survived. The remaining 10% died within several days, apparently from irradiation-induced diarrhoea. Assuming 10% of injected cells were haematopoietic precursors, 104such cells were grafted, approximately equal to 1.5% of the total numbers calculated to exist in recipients.
|A. Numbers of colonized recipients|
|Duration post-grafting (days)||Percentage of recipients with donor markers|
|8||0||3 (9)||3 (8)||12 (1)|
|16||0||0 (1)||2 (10)||16 (7)|
|32||0||0 (0)||0 (0)||18 (18)|
|300||0||0 (0)||0 (0)||18 (18)|
|Recipients with donor markers: haemoglobin without brackets, lymphopoietic GPI in brackets. Levels assessed by densitometry.|
|B. Expression of donor markers and period of recipient survival|
|Nucleated cells injected (×104)||Donor cells in culture (days)||No. of recipients with donor markers||Recipient survival (days)|
|C. Haematological parameters in 129-WvWv anaemic mice after grafting with C57Bl/10 blastocyst outgrowths cultured for 3 or 4 days|
|Time after graft (days)||Haemoglobin (g/100 ml)||Erythrocytes (×109 per ml)||Leukocytes (×106 per ml)|
Most recipients survived to old ages. Autopsies revealed donor chromosome markers in bone marrow, and continued expression of donor markers for erythropoiesis and lymphopoiesis. Control irradiated, non-grafted mice died within 12–14 days as their levels of haemoglobin and GPI declined to very low levels ( Table 10 ). Among other controls, cells from blastocysts cultured for <3 days did not colonize recipients, and ES cell lines prepared according to standard methods also failed to colonize any recipient. Skin grafts involving the donor strain C57Bl/10 were rejected 12–14 days after grafting onto recipients expressing donor markers, rejection time being similar to that in normal untreated recipients.
Section 7.2. Use of cultured blastocyst stem cells to repair non-irradiated anaemic recipients
Blastocyst stem cells were next tested for their ability to repair inherited anaemia in non-irradiated recipients. Recipients from strain 129-WvWvmice, homozygous for an inherited macrocytic anaemia (WvWv), were grafted via tail vein within 1–2 days of weaning, i.e. at ages 21–22 days (Hollands, 1988a). Early injections were needed, since 90% of these homozygous mice die within 2–3 days of weaning through their inherited anaemia.
Between 4.9 and 10.5 × 104grafted cells were equally effective in colonizing recipients (Hollands, 1988a). Donor haemoglobin appeared in 90% of these mice 24–48 h after injection (Table 10 and Table 11). Using previous methods, they represented 10% of total haematopoietic cells in recipients, reaching virtually 100% by day 16. Likewise, donor GPI appeared in the same recipients, indicating that donor lymphocytes were present by days 3–4 in 90% of recipients, rising to virtually 100% by day 16. Levels of haemoglobin and erythrocyte counts in injectedWvWvmice returned to normal, as their characteristic macrocytosis completely disappeared ( Table 11 ). The remaining 10% of recipient mice died within 2–3 days, as in controls, and did not display donor markers. Control non-injected mice died after 2–3 days.
|A. Data on grafting WvWv recipients|
|No. recipients||No. nucleated cells ×106||Age of donor embryos (days)||Donor Hb/GPI||Survival (days)|
|B. Minimal number of effective cells|
|No. recipients||No. injected cells ×106||Donor Hb/GPI||Survival (days)|
|C. Gene markers and the degree of colonization of injected recipients (values are numbers of mice)|
|Interval post-grafting (days)||Degree of colonisation|
Among controls, cells isolated from blastocysts grown for <3 days did not colonize recipients. ES or EC cells prepared according to Solter and Damjanov (1979), Evans and Kaufman (1981) and Martin (1981) failed to colonize haematopoietic systems ofWvWvmice, just as they failed with irradiated Balb/c mice. No markers were identified in any recipient. This result contrasts with the ability of freshly grown blastocyst stem cells to colonize intact animals without need for X‐irradiation.
Hollands (1988b,c) repeated this study using intact mice carrying other forms of anemia. These included 129-WvWv,Wsha less severe anaemia where mice lived normal life durations,Sl/+(Steel anaemia),Slp(Steel/Peru anaemia), and athymicnudemice. Injections were made at 1–2 days post weaning inWv mice, and at 5–6 weeks of life or slightly later inWshrecipients and the other strains. Among the anaemic mice,Wvhad macrocytic anaemia, and the others had milder forms that did not threaten life. Donors were MF1 outbred mice and strain C57Bl/10 inbred mice.
Stem cells prepared from day 5 embryos failed to colonize when injected intoWvWvmice, as earlier. All 30 of these mice injected with cells from cultured blastocysts displayed donor markers and lived for >600 days ( Table 12 ). Minimal numbers of cells for successful grafting and the degree of colonization with increased post-graft intervals are also shown there. By day 6, donor haemoglobin and GPI had virtually reached 100%. Bone marrow infusions toWvWvrecipients failed. Approximately 30% of intact animals were colonized in strains carryingWsh,Sl/+andSl,pwhich do not succumb to anaemia.
|Time (h) post-insemination||Embryo 1||Embryo 2||Comments|
Some HCG secretion
|216||Attached to plastic||−||Some HCG secretion|
|Fibroblasts from ICM a||−||−|
|288||Extensive trophoblast outgrowth||−||High HCG secretion from embryo 1|
a ICM = inner cell mass.
Section 8. Migratory pathways of stem cells
Stem cells from early post-implantation embryos and blastocysts migrated through liver before any colonized bone marrow. They followed the fetal pathway in adult recipients, finally entering bone marrow (Hollands and Edwards, 1986; Hollands, 1987, 1988b,c).
T6 cells entered liver within 24–48 h of grafting. Their cell divisions there ended soon afterwards. Some may have been stem hepatocytes, or even precursors of other tissues. Many if not all then colonized bone marrow, numbers increasing rapidly (Table 7 and Table 9). Some entered spleen. It is clearly essential to discover if injected stem cells were derived from several distinct tissue precursors. Their potential for colonizing other organs was not checked.
Colonization of liver and bone marrow and restoration of haematopoiesis were not species specific. Rat stem cells were equally effective in migrating to recipient mouse liver, identified by numerous rat mitoses, and then to bone marrow. They were also equally effective in sustaining a normal life period in mouse recipients.
No evidence of neoplasia or inflammation was noted in recipient mice throughout the whole of this work. The potential of embryo stem cells for colonization was now very clear ( Figure 4 ).
Section 9. Preliminary studies with human stem cells
Full progress with IVF in Bourn Hall in the early 1980s was accompanied by an increasing crescendo of ethical debates. Contentious issues covered the right to fertilize human eggsin vitro, using human embryos for research, risking cloning, using donor oocytes, establishing surrogate pregnancies, and risks with embryo cryopreservation among other matters.
During IVF, a few ‘spare’ human blastocyts were seemingly of no value for replacement into their mothers since their chances of implantation were low. We decided to use some of them in attempts to produce human embryo stem cell linesin vitro. Proposals to use human embryonic stem cells to cure diseased organs in human recipients were first made in 1971 (Edwards, 1971, 1980), following earlier discussions with John Paul and Robin Cole. Guaranteeing the production of highest quality embryo stem cells would ultimately require human blastocysts with fully competent inner cell masses capable of producing full-term babies. Human blastocysts had been grown to day 5, and then to day 9, with excellent inner cell masses of embryonic discs respectively, signifying it was time to initiate attempts at using ‘spare’ human blastocysts for making embryo stem cells (Steptoeet al., 1971; Edwards and Surani, 1978). The birth of more than 100 IVF children from Bourn Hall by the early 1980s using IVF now provided the opportunity to begin. Previous experience with grafting early stem cells was zero, although grafts of tissues from mid-term human fetuses were known to repair various human organs as discussed earlier. Graft rejection limited their value, blighting attempts to alleviate adult diabetes by grafting cells from fetal pancreas (Sutherland, 1981), and exchanges between identical twins seemed the only option (Feferet al., 1982). Nevertheless, by the later 1980s, Hollands (1988b,c) had shown how mouse embryo stem cells avoided graft rejection, and other such indications were being reviewed (Edwards, 1992).
Six human eggs, fertilizedin vitroand cultured in Earle's medium reinforced with pyruvate and 15% v/v maternal serum, were used in attempts to prepare human stem cells (Fishelet al., 1984). Assays for βHCG on ‘spent’ culture media measured trophoblast activity. Two embryos secreted this hormone into medium from 120 and 170 h post-insemination, when levels rose considerably. Two zona-free blastocysts expanded and attached to the plastic culture dish at 189 and 216 h post-insemination respectively. Embryo structure was lost as trophoblast migrated from one embryo, which collapsed at 246 h ( Table 12 ). Extensive fibroblastic cells migrated from inner cell mass about this time, among outgrowths of trophoblast before all tissues regressed at 317 h. The second embryo collapsed at 197 h and became degenerate.
Ethical problems emerged with plans to extend this work, concerning sources of blastocysts for stem cells and their use for this purpose. Requests for parents' permission or gaining consent to grow embryos to blastocysts for this purpose proved to be complex. Further ethical issues emerged over the fate of remaining embryos when the best had been transferred to their mothers. No means were available to identify embryos with very low chances of implantation that could be used in research, and those capable of implantation and cryopreservation for their parents. All embryos were therefore cryopreserved for parents' later use, a joint decision taken by Bourn Hall staff and the Ethical Committee. Stem cell and other research then effectively ended in 1984, this ban extending until 10 years later, when terms of consent allowed parents to donate their cryopreserved embryos for research.
Section 10. Embryo stem cells 1963–1992
Section 10.1. Assumptions and expectations
Looking over this astonishing period of the discovery and work on embryo stem cells, some previous assumptions have been dispelled and others confirmed. Work on rabbit stem cells in the 1960s dispelled a belief that somatic cells were restricted to ca. 50 generationsin vitro(Hayflick and Moorhead, 1961). In those days, brain, myocardium and other tissue were thought to lack stem cells, another idea dispelled today. Some early assumptions proved to be correct, e.g. that donor human embryos must have reached Carnegie Stage 5in vitro, i.e. 7–10 days of growth, when the embryonic yolk sac is initiated (Hollands, 1991). Earlier hopes that that embryo stem cells may avoid graft rejection in histoincompatible recipients were also confirmed (Hollands, 1988b,c).
Correct predictions also included the wide potential of stem cells and the potential of cloning to produce human stem cells compatible with recipients. Their clinical benefits were first briefly raised 30 years ago (Edwards, 1971), and during an address to the Vatican Academy of Science (Edwards, 1984): ‘This fanciful use of embryonic stem cells for grafting into adults is perhaps the ultimate application of in-vitro fertilization’. Reviews, book chapters, unpublished lectures, and debates (e.g. Edwards, 1982) discussing the ethics of IVF stressed repeatedly how embryo stem cells ‘could offer a unique advantage, because graft rejection could be completely avoided … by experimental methods to tailor embryos to suit a particular recipient. Replica tissues without any histoincompatibility for a woman could be formed by inducing parthenogenesis in her oocytes … and androgenesis would offer similar benefits for men. Tissues compatible with an adult host might also be obtained through cloning’. An appeal was made for ethical consideration of using embryonic cells for organ repair (Edwards, 1982).
A book published in 1992 set the scene on embryo and fetal stem cell lines for studies in the later 1990s (Edwards, 1992). Its chapters included procuring embryonic and fetal tissues and the ethics of fetal tissue transplantation (Polkinghorne, 1992; Wong, 1992). Promising systems included transferring in-utero fetal liver as a source of haemopoietic donor tissues for unrelated fetuses of various ages inheriting immunodeficiency diseases without incurring rejection (Touraine, 1992), grafts of fetal brain (Saueret al., 1992), fetal pancreas (Tuch, 1992), cornea and gonadal tissue. The use of coelocentesis for donating tissue to first-trimester human fetuses was raised 3 years later (Edwardset al., 1995). Some clinical conditions were assumed to need full colonization, e.g. haemopoietic tissues after cancer therapy, whereas partial colonization could suffice for others, e.g. sickle-cell anaemia. Since donor embryo stem cells migrated through liver to bone marrow and spleen, other types of stem cells may also migrate via fetal pathways to other tissues after injection into recipients' tail veins.
Very few embryo stem cells seem to be needed for successful grafting. Hollands may have injected 103or less into mouse recipients, and a very rapid cell cycle must have sustained their high multiplication. This is not too surprising, since as few as 50 bone marrow precursor cells colonize thymus glands and haematopoietic systems in lethally X‐irradiated recipient adult mice (Williamset al., 1984; Spangrudeet al., 1988; Joneset al., 1989). They apparently colonize slowly but permanently, whereas committed precursor cells colonize quickly but less permanently. Hollands' studies implied that cultured embryo stem cells from day 6 mouse blastocysts or early implanted embryos colonized fast and persisted well, whereas those taken at day 5 failed to colonize. Haemopoietic potential must develop between these ages.
New surprises may be in store, for example on the origins of embryo and tissue stem cells ( Figure 5 ). Are all stem cells in various tissues related, arising from a common source? Do they have a shared origin with primordial germ cells, each inheriting immense propensities for widespread tissue migration? Embryo stem cells prepared by Hollands might resemble inner cell mass and differ from ES and bone marrow cells which failed to colonize recipient bone marrow (Hollands, 1988a,b,c). Embryo stem cells have very wide potencies, perhaps unmatched even by mesenchyme, known today to be widely multipotent. Embryo stem cells, and perhaps mesenchyme, might indeed establish unexpectedly wide forms of organ chimaerism in adult recipients given mesenchyme and newborns given cord blood. They may now be complex chimaeras, including an elder sibling saved by cord blood stem cells from a ‘designer baby’ (Verlinskyet al., 2000).
Section 11. Ethical surprises and dilemmas
Recent ethical decisions mirror those emerging over preparing human stem cell lines in Bourn Hall. The same ethics engulfed the US president and government, and now the German government (Ludwig, 2002), who opposed using human blastocysts for making embryo stem cells in governmental laboratories. They decided instead to purchase them from private clinics or from abroad. This is a vain attempt to retain a clear conscience, even as they assure the public they had not sanctioned this use of human embryos. Such seriously flawed ethics, to put it mildly, fail to shift ethical responsibility from those who commission them to those who made them. Commissioning them is far less honourable than making them. Avoiding ethical responsibility in this way still deprives parents of their embryos and reduces human embryos to the status of research items. Donor clinics may be located in countries with low or no standards of informed consent or laboratory practice. Purchasing stem cell lines merely encourages providers to supply more and more, extending the ethical compromise.
A similar ethical dilemma has struck the UK government. Desiring to avoid graft rejection in recipients, it sanctioned legislation permitting ‘therapeutic’ human cloning. Yet transferring adult nuclei into oocytes is associated with endless embryological disasters in several mammals. Not only this, since human embryos inherit specific disasters preventing 80% of them from implantingin vivo. Cloned human embryo stem cells will inherit both sets of disasters, risking the health of sick adult recipients. Ironically, on virtually the same day this legislation was accepted, new research confirmed again the capacity of embryo stem cells to avoid graft rejection in fetal and adult recipients (Edwards, 1971; Touraine, 1983, 1992; Hollands, 1991; Fandrichet al., 2002). The complicated legislative ethics of therapeutic human cloning to avoid rejection could well have been avoided if more work had been done on how embryo stem cells do it naturally. And every study published on therapeutic cloning aids those clinicians and scientists wishing to clone human beings despite the risks.
Section 12. Summary
Introducing IVF, preimplantation genetic diagnosis and embryo stem cell lines has been an exhilarating experience. These three stunning approaches to averting or mending human disorders were invented through collaboration with many teachers, colleagues and students. I am still thrilled when, in today's world of molecular biology and reconstructive surgery, tissues are switched to desired pathways on three-dimensional biodegradable meshes coated with specific substrates. Meshes shaped like human organs such as ears or thumbs promise immense human benefits and even more when allied with stem cells.
This paper has described work on embryo stem cells since its inception in the early 1960s. The astonishing properties of embryo stem cells for proliferation, colonization and differentiation enables them to colonize virtually all organs, whether placed in blastocoelic cavities or injected into tail veins. Their enormous potential led me to near-euphoria in the 1980s about clinical prospects, appreciated then by very few other investigators. The same delighted responses are notable today as new investigators first appreciate these astonishing properties.
The amazing immortality of embryo stem cells, first identified in 1963, overturned existing beliefs on limited lifespans in mammalian cell lines, or that stem cells were absent from brain and myocardium. Immense proliferative and colonizing characteristics of stem cells promise repairs to deficient human organs, avoiding graft rejection, migrating via fetal pathways and target damaged tissues without invoking inflammation or cancer, or any need for surgery. UK legislation to permit therapeutic cloning of stem cells will doubtless be copied elsewhere. The tragedy is that they could be in clinical use today if initial proposals had not been cast aside with incredulity, even disdain.Naturerejected a foundation paper in the 1980s, questioning how so few stem cells could colonize major organs.Scienceaccepted one paper, but with clear hostility and veiled references to flawed ethics. And today, this early foundation work on stem cells is neither recognized nor quoted by current workers still behind in many aspects and patenting their current work. This narrow vision was exemplified by one professional commentator, ecstatically proclaiming how adult cloning and embryo stem cells were manifestations of the ‘last five years of the twentieth century’ (Aldhous, 2001). Just how wrong can he get?
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Editor, Reproductive BioMedicine Online, Duck End Farm, Dry Drayton, Cambridge CB3 8DB, UK
# Original publication: Edwards, R.G., 2002. Personal pathways to embryonic stem cells. Reprod. BioMed. Online 4, 263–278.
1 Paper based on contribution presented at the Alpha meeting in New York, USA, September 2001.
© 2010 Reproductive Healthcare Ltd., Published by Elsevier B.V.