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STEM CELLS IN THE NEWS |
a Executive Director, Cell Therapy R & D Head, Hematopoiesis Department Holland Laboratory, American Red Cross hawleyr{at}usa.redcross.org
b Holland Laboratory, American Red Cross sobieski{at}usa.redcross.org
"Contrariwise," continued Tweedledee, "if it was so, it might be; and if it were so, it would be; but as it isn’t, it ain’t. That’s logic."Lewis Carroll, "Through the Looking-Glass"
WORLD’S FIRST XENO-FREE HUMAN EMBRYONIC STEM CELLS
Researchers at the National University of Singapore reported in the online version of Nature Biotechnology on August 5 that they had created the world’s first human embryonic stem (ES) cell line grown entirely without exposure to mouse feeder layers or other nonhuman proteins. The new human cell line established by Richards et al. [1] from the inner cell mass stage of development, which at time of publication had been maintained for 50 generations without differentiation and continued to display a normal karyotype, expressed the ES cell-associated transcription factor Oct-4 and cell surface markers SSEA-3, SSEA-4, Tra-1-60, and GCTM-2, and tested positive for alkaline phosphatase activity. Upon injection into severe combined immunodeficient (SCID) mice, the cells formed teratomas containing tissues from all three germ layers, confirming their pluripotentiality. By using human feeder layers and human serum to support the derivation and growth of human ES cells, the investigators noted that they eliminated the risk of transmission of animal pathogens to the cells. Human feeder-supported ES cells should thus face fewer hurdles to U.S. Food and Drug Administration approval for any potential clinical use than the 78 existing human ES cell lines listed on the U.S. National Institutes of Health Stem Cell Registry website (http://escr.nih.gov/), the derivatives of which would be considered xenotransplantation products. The new results do little to assuage the significant ethical concerns already associated with this line of research, however, since two of the three human feeder layers used in the study were derived from fetal cells obtained from human abortuses.
PLURIPOTENT ADULT MESENCHYMAL STEM CELLS IN BONE MARROW, MUSCLE, AND BRAIN
Jiang et al. [2] reported in the July 4 issue of Nature that they established pluripotent adult mesenchymal cells—termed multipotent adult progenitor cells (MAPCs)—from bone marrow of adult mice and rats, which could be propagated for more than 120 population doublings in culture. MAPCs were shown to express Oct-4 (albeit at low levels), which, besides ES cells, is normally restricted in its expression to the inner cell mass and epiblast of the embryo and to germ cells [3]. More important, the authors demonstrated that MAPCs could be induced to differentiate in vitro and in vivo into a variety of cell types of mesodermal, neuroectodermal, and endodermal origin (i.e., representatives of all three germ layers). To determine the fate of MAPCs in the developing embryo, the authors microinjected either a single murine MAPC or 10 to 12 MAPCs into 3.5-day-old blastocysts, which were transferred to foster mothers and allowed to progress to term. The resulting animals showed no unusual characteristics. Chimerism-testing by tail snip analysis at 4 weeks of age demonstrated that 80% of blastocysts injected with multiple MAPCs and 33% of those injected with a single MAPC gave rise to animals with chimeric cellular composition, ranging from 0.1%-45%. Animals were killed at 6 to 20 weeks and analyzed by either thin section staining for presence of MAPC-derived cells or by analysis of organs. MAPC-derived cells were found in many tissues, including brain, lung, myocardium, skeletal muscle, liver, intestine, bone marrow, kidney, spleen, and blood. As an additional test of the developmental repertoire of MAPCs, the investigators infused the cells intravenously into non-irradiated or sublethally irradiated 6- to 8-week-old nonobese diabetic (NOD)/SCID mice and looked for evidence of engraftment 4 to 24 weeks after transplantation. The researchers observed engraftment in bone marrow, spleen, blood, and epithelium of lung, liver, and intestine of all recipient animals. No engraftment was observed in skeletal or cardiac muscle, skin, kidney, or brain. The authors noted that they did not detect any MAPC-derived tumors in any of the experiments. On the other hand, a host-derived lymphoma that arose in one NOD/SCID recipient contained 40% MAPC-derived endothelial cells within the tumor vasculature. In a follow-up article published in the August issue of Experimental Hematology, Verfaillie and colleagues [4] reported that cells with MAPC characteristics could also be established from muscle and brain tissues obtained from mice. These amazing findings with murine and rat MAPCs extend earlier studies of human MAPCs by Verfaillie’s group [5–7] and are certain to be heralded by opponents of human ES cell research. Interestingly, while media attention and a major impetus behind the work concerns potential future therapeutic applications of MAPCs, the discovery of these apparently pluripotent adult cells may have provided a solution to a longstanding research problem. Stable rat ES cell lines have been notoriously difficult to derive from preimplantation rat embryos [8]. If MAPCs are truly totipotent and can contribute to the germline, gene-targeted rat MAPCs could be used as an alternative to create new chimeric rat models of human disease [9].
SMARTER WAYS TO MAKE BRAIN CELLS
In a companion article to that of Jiang et al. in the July 4 issue of Nature, McKay and colleagues [10] described a method to increase the efficiency of generating dopamine neurons—those destroyed in Parkinson’s disease—from murine ES cells. The investigators established gene-modified ES cell lines that stably express the nuclear receptor related-1 (Nurr1) gene, which codes for a transcription factor required for differentiation of midbrain precursor cells into dopamine-producing neurons. Nurr1-ES cells were then treated with fibroblast growth factor-8 and sonic hedgehog, developmentally relevant signals generated in vivo by the isthmic organizer, to induce neurogenesis to tyrosine hydroxylase (TH)-producing cells. The 78% of Nurr1-ES cells that became TH+ cells were found to exhibit many molecular, morphological, and functional features of midbrain dopamine neurons. The researchers then assessed the ability of differentiated Nurr1-ES cells to survive, integrate, and function in host animals; rats that had been treated with 6-hydroxy dopamine, which kills dopamine neurons, providing a model of Parkinson’s disease. Upon morphological analysis, the animals displayed successful engraftment of cells with the mouse-specific surface antigen M2. The engrafted cells had neuronal characteristics and exhibited evidence of synapse formation. Importantly, the number of engrafted cells remained stable between 4 and 8 weeks. This observation contrasts with results of studies of undifferentiated ES cells, where the cells frequently continue to divide following engraftment giving rise to teratomas. The host animals showed improved neurological characteristics when evaluated with a variety of tests compared to sham-operated animals. The improvement in behavioral responses indicated that the engrafted neurons restored some level of dopamine-related function to the animals.
A complementary study by Song et al. published earlier this year (in the May 2 issue of Nature) [11] described the differentiation of neural stem cells isolated from the hippocampus of an adult rat into neurons through the influence of factors produced by astrocytes. The investigators first cultured fibroblast growth factor-2-dependent adult neural stem cells, which had been labeled with a green fluorescent protein transgene to allow tracking of cellular progeny, under serum-free conditions on feeder layers of primary neurons and astrocytes derived from neonatal hippocampus. This procedure gave rise to significant numbers of neurons as well as to some oligodendrocytes and astrocytes. Further studies showed that primary hippocampal astrocytes alone are capable of inducing neurogenesis from these adult neural stem cells, and that this activity was associated with both diffusible and membrane-bound astrocyte factors. The increased net neurogenesis did not appear to be simply the result of improved neuronal survival. Further studies and mathematical analysis of the proliferation, differentiation, and death of cultured neuronal cells showed that hippocampal astrocytes increased both proliferation and neuronal fate commitment of the adult neural stem cells. The investigators subsequently demonstrated that astrocytes derived from adult hippocampus also promoted neurogenesis although they were only about half as effective as those derived from neonatal hippocampus. Finally, while adult hippocampal astrocytes promoted commitment of adult neural stem cells to become neurons, astrocytes derived from adult spinal cord did not. The findings raise the notion that hippocampal astrocytes may play a unique regulatory role in promoting the development of new neurons in the mature central nervous system—an observation that could perhaps be exploited in future stem cell-based regenerative therapies for treatment of neurological disorders such as Parkinson’s disease.
NUCLEAR TRANSPLANT ES CELLS FOR TISSUE BIOENGENEERING
The ability to readily produce histocompatible cells for tissue regeneration would circumvent one of the major complications associated with allotransplantation [12]. Although proteins encoded by genomic DNA are the primary cause of histocompatibility problems, the translation products of mitochondrial DNA are clearly a source of minor histocompatibility antigens. In the July issue of Nature Biotechnology, Lanza et al. [13] described the use of nuclear transplantation technology (therapeutic cloning) to study the implications of immune response to mitochondrial antigens in tissue replacement therapies. The group transferred nuclei from adult bovine dermal fibroblasts into enucleated bovine oocytes, and implanted cloned cardiac and skeletal muscle cells and renal cells obtained from early-stage fetuses back into nuclear donor animals. The mitochondrial DNA of the nuclear donor differed from that of the oocyte donor, and two of the resulting amino acid substitutions were expected to be immunogenic. Cardiac and skeletal tissues were collected from 5- to 6-week-old fetuses and renal tissue from 7- to 8-week-old fetuses. Cells from the collected tissues were isolated, expanded in vitro, and applied to polyglycolic acid polymer or polycarbonate membrane delivery vehicles. The membrane containing the renal cells was formed into cylinders with a sealed reservoir at one end. The cell-polymer constructs were then subcutaneously implanted into the flank of the steer that provided the transferred nuclei. Similar constructs containing cells from allogeneic fetuses were implanted into the same animal as a control. Constructs were retrieved at 6 and 12 weeks following implantation and analyzed for cellular development and histocompatibility with the recipient. In the case of the muscle cells, a second set of constructs from the same donor was transplanted for an additional 12 weeks after retrieval of the first set of implants. Muscle explants from the cloned cells showed organization into muscle bundles and extensive vascularization, while explants from the control cells showed lack of tissue development as well as evidence of inflammation and immune rejection. Semiquantitative reverse transcription-polymerase chain reaction and Western blot analysis indicated higher levels of muscle-specific mRNA and proteins in the cloned muscle tissues than in the allogeneic tissue grafts. The presence of allogeneic mitochondrial antigens in cells derived by nuclear transfer did not appear to significantly impede the development of the muscle tissues. Of particular note, the retrieved renal constructs derived from cloned cells, which also showed significant vascularization, appeared to have self-assembled into glomeruli and tubule-like structures. The tubules were observed to contain yellow fluid that showed evidence of unidirectional concentration of urea nitrogen. Delayed-type hypersensitivity analysis and enzyme-linked immunospot assay estimates of interferon-
-secreting T cells suggested that there was no rejection response specific for the nuclear transfer-generated cells. Although further studies will be necessary to formally demonstrate that the kidney structures formed were indeed functional, the results are a major proof-of-principle step toward this goal. Nevertheless, as acknowledged by the authors, the strategy could not be applied in humans, as generating renal units from similarly derived fetal tissue is not an ethically acceptable option.
PANCREATIC ISLET-LIKE STRUCTURES FROM HEPATIC OVAL CELLS
While biologists continue to debate the validity of somatic stem cell plasticity [14,15], Yang et al. reported in the June 11 issue of the Proceedings of the National Academy of Sciences [16] the apparent in vitro transdifferentiation of adult rat hepatic oval stem cells into pancreatic endocrine hormone-producing cells. The researchers induced the unconventional differentiation by removing leukemia inhibitory factor (an inhibitor of stem cell differentiation) and culturing the cells in medium containing high concentrations of glucose. They obtained differentiated cells that formed islet cell-like three-dimensional clusters, expressed pancreatic islet cell differentiation-related transcripts, and produced islet-specific hormones such as insulin, glucagons, and pancreatic polypeptide. The investigators conducted a pilot study in vivo by transplanting the transdifferentiated cells into NOD/SCID mice, one of which showed reversal of hyperglycemia 10 days after implantation of 200 islet-like clusters divided between the renal subcapsular space and a subcutaneous site. Recipient mice that received only 30 clusters implanted in the subcapsular space did not show improvement. Previous observations have suggested that hepatic oval and other cells may arise from a cell population originating in, or associated with, the bone marrow [7,17,18]. The authors thus raised the possibility that in the future autologous bone marrow stem/progenitor cells could be collected and differentiated in vitro into insulin-producing islet-like cells that could be used to treat diabetes in human patients.
HOMOZYGOUS MUTANT MICE PRODUCED BY TETRAPLOID EMBRYO COMPLEMENTATION
Conventional production of genetically engineered homozygous mutant mice is time consuming and expensive. In the May issue of Nature Biotechnology, Eggan et al. [19] described an abbreviated method of generating homozygous mutant mice by crossing male (XY) and female (X0) mice derived from the same genetically manipulated ES cell line via tetraploid embryo complementation. The researchers observed that about 2% of subclones from a variety of male mouse ES cell lines spontaneously lost the Y chromosome, and were able to successfully produce X0 female mice from X0 ES cells via the tetraploid embryo complementation protocol. XY ES cells from the same line gave rise to male offspring using the same procedures. This enabled the investigators to produce both male and female target-chimeric mice from genetically targeted ES cells and then intercross the resulting cloned mice to obtain progeny with XY, X0, and XX karyotypes. The offspring of the cross showed normal Mendelian assortment of the targeted trait, allowing the authors to obtain target-homozygous mutant mice in a single intercross. The researchers were also able to show that repeated rounds of in vitro ES cell genetic manipulation to introduce more than one target mutation did not affect the ability to derive male and female multimutant clones. This technique enables researchers to obtain offspring homozygous at a variety of target sites from crosses of clones containing the desired sequences. Also noteworthy was the observation that although ES cells lines accumulate karyotypic abnormalities during repeated passaging, clones generated from the cells via tetraploid embryo complementation carried a normal chromosomal complement, possibly because only cells of normal karyotype contribute to embryonic development. The only caveat to the strategy described is that F1 ES cells were used. The offspring of F1 ES cell-tetraploid mutant mice are a genetically heterogeneous F2 population. As discussed by the investigators, if mutant animals with an inbred genetic background are required, substantial backcrossing would be necessary, abrogating many benefits of the new methodology.
DISCLAIMER
Any views and opinions expressed herein are those of the authors. They do not necessarily reflect the policies or position of the American Red Cross.
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