Topic of the Month Highlights of the 2004 Keystone By
Suzanne Kadereit For 5 days in late January, stem cell researchers from around the world gathered at Keystone Resort in Colorado to ski and discuss stem cells.
Stem Cell Plasticity The debate on stem cell plasticity has regained momentum. Some convincing results suggest that there is plasticity, but that the frequency is so low that its application for therapy would be particularly difficult to develop. Other results suggest that there may be very primitive ES cell-like cells in the bone marrow and the skin. What are the mechanisms and cues to activate the cells to (trans)-differentiate? One emerging concept appears to be that injury or tissue regeneration are required to induce the cell fate change. During this new and improved round of debate any claimant of plasticity has to demonstrate that the results are not confounded by cell fusion. Dr. Krause's group addressed the issue by transplanting bone marrow cells from a male mouse that expresses the b-gal gene, driven by the actin promoter, flanked by a stop codon and lox sites and followed by EFGP (Z/EG construct), into a lethally irradiated female mouse that ubiquitously expresses Cre-recombinase. Upon fusion of the male transplanted cells and host female cells, the fusion products would not only express the Y chromosome, but would also excise the b-gal gene and constitutively express GFP. Cells that did not fuse would express the Y chromosome and the b-gal gene. The excisability of the Z/EG construct was verified with cell fusion in vitro. The efficacy of the cre-recombinase expression and activity was tested by infecting the Cre mice with an adenovirus construct that expresses a construct similar to the Z/EG construct. Even the accessibility of the Z/EG construct within the cellular chromatin was assessed by transfecting the cells with a constitutively expressing Cre-recombinase construct. All three approaches confirmed that the assay system worked. Upon transplanting male bone marrow cells into female mice, the group could not find any indication of fusion (cells expressing GFP), but they did find pneumocytes that expressed b-gal and had Y chromosomes, suggesting that transplanted bone marrow cells differentiated into pneumocytes. In the muscle of lethally irradiated mice that had their bone marrow
reconstituted with transplantation of a single cell (SP cell), it
would appear that bone marrow-derived cells first become satellite
cell-like cells and occupy the niche in the damaged or (re)generating
muscle, and in a second step help to regenerate mature muscle fibers
in response to environmental cues. However, frequency is low, as
was shown by Dr. Blau's group. Frequency increases over time when
the animals are subjected to exercise, and increases dramatically
when entire muscle fibers are removed and neighboring muscle tissue
has to compensate for the loss of function. However, in the absence
of any injury or irradiation, in the parabiotic model, no donor
cells could be detected. The same group also showed that in the
case of fusion, rather than transdifferentiation, it would seem
that to a certain extent reprogramming of the fused nuclei takes
place. In the murine system, injected bone marrow-derived cells
that express GFP under a Purkinje neuron-specific promoter, L7-GFP,
did fuse with resident adult Purkinje neurons as detected by heterokaryon
formation, but also expressed the L7-GFP transgene. But, in the cases where transdifferentiation has been demonstrated, what is this elusive cell that can generate different tissues? Could it be the mesenchymal stem cell (MSC) or the MAPC (multipotent adult progenitor cell)? The latter expresses ES-specific genes. In-depth comparison of the two cell types by gene array is currently underway. In transplantation experiments by Dr. Verfaillie's group, single MAPCs injected into mouse blastocysts resulted in 1/3 of the animals being chimeric, results similar to those with injected ES cells. However, with injection of over 10e5 MAPCs into newborns, where tissue development is still ongoing, MAPC-derived cells could only be found in extremely low numbers in resulting adult tissues. Higher rates of engraftment were observed if the cells were injected into E16 old or newborn MPS-1 mice (model for muccupolysacharidosis), resulting in amelioration of the phenotype. Another cell of interest was reported in the skin. Dr. Miller's group identified a cell in the hair follicle bulge that could also be isolated from skin that has no hair, the Skin-derived Precursor (SKP), which, when exposed to only two growth factors, forms spheres in culture. These spheres express numerous embryonic transcription factors and share many attributes with neural crest stem cells, and, at the single cell level, can give raise to neuronal and mesodermal progeny. While it is still unclear which cell in the bone marrow transdifferentiates, it does not seem to be the HSC, as shown by single-cell transplant experiments. When individual HSC were transplanted by Dr. Weissman's group into ablated mice, only hematopoietic tissue regenerated, in absence of injury. And even in the presence of injury, the extent of differentiation into other types of cells was low. A study presented by Dr. Stadtfeld confirmed this finding. When crossing mice expressing Cre recombinase under the pan-hematopoietic promoter of Vav with Rosa26-lacZ and Rosa26-EYFP mice, all hematopoietic cells, HSC included, were found labeled in the F1 generation. Any labeled, non-hematopoietic cell in these offspring mice would be indicative of a possible switch from hematopoietic fate to non-hematopoietic fate during development. When tissues of these mice were examined, all tissues tested were found negative, with the exception of skeletal muscle where a high proportion of cells were labeled. But all these sophisticated studies beg the question: does plasticity really happen as a part of normal physiology in the adult? There is the interesting phenomenon of fetal/maternal microchimerism, where fetal cells pass into the maternal circulation, and vice-versa. Maternal cells can persist into adulthood in immunocompetent offspring, and Y chromosome positive cells have been reported years later in women who had sons, and may possibly contribute to certain autoimmune diseases in women. Fetal-origin cells were found to have reconstituted liver damage, as well as thyroid damage, and the reconstituting cells expressed organ-specific markers. When Dr. Bianchi's group assessed fetal cell transmission in a GFP-mouse model (female wt x male tgGFP), it was shown that all pregnant mice had microchimerism in most tissues studied, with the lung being the most affected. When retired breeders females were challenged with chemical injury to the liver, microchimerism was increased in the livers of the challenged mice, suggesting that cells of fetal origin, termed Pregnancy Associated Progenitor Cell (PAPC) have a certain plasticity and may aid tissue recovery. On a final note, proponents of transplanting a patient's own bone
marrow cells to reconstitute damaged heart tissue may be interested
to know that one group has shown data suggesting a functional impairment
in bone marrow cells of patients with chronic ischemic heart disease.
This new finding and the documented diminished self-renewal capacity
of old hematopoietic stem cells, render therapeutic success of transplantation
of own bone marrow in older heart patients, the majority of those
patients, questionable. Much more research into the phenomenon of
plasticity and the molecular mechanism regulating stem cells is
needed, before adult stem cells can be used on a larger clinical
scale. Molecular Characterization of Stem Cells Lately, with the advent of gene array technology, molecular characterization of stem cells has taken significant leaps. It is now 'de rigueur', as it has become clear that simple surface characterization is not sufficient. Other means are needed, such as defined transcription factors or gene expression program, characteristic of stem cell subpopulation, developmental intermediate stages, or lineage specification. Not surprisingly then, reports on expression profiling of stem cells by gene microarrays abounded at the meeting. Hipp et al. reported comparison of ES cells (derived from parthenogenotes of Macaca fascicularis eggs), with their progeny. Their data mirrored previously reported gene array data. Venezia et al. reported comparison of quiescent adult HSC, HSC induced to proliferate by 5-fluorouracil, and naturally cycling fetal liver HSC. The group established molecular signatures for quiescent and proliferating stem cells, and identified two genes that could potentially be new markers for proliferating stem cells. Wang et al. reported a meta analysis, performed on human and mouse ES cells as well as on zebrafish embryos, in an effort to determine genes responsive to Oct-4, and thus define core regulatory pathways for stem cell identity. One such gene, draculin, has been identified comparing mouse and zebrafish. Its promoter contains multiple upstream binding sites for Oct-4. An additional factor was identified by Yamanaka et al., who analyzed expression gene profiles of ES cells. ECAT3 (ES cell associated transcript 3), whose expression is controlled by Oct-4 and Sox-2, and which could be involved in protein degradation. Dr Nishikawa's group, after carefully defining culture conditions to steer ES cells towards mesodermal fate, analyzed 30 intermediate stages by microarrays and compiled a data base. Dr. Orwig's group used gene array to compare mouse testes depleted of stem cells by heat and chemotherapy, with unmanipulated testes, and was able to narrow down the stem cell activity to a CCR2 negative cell. Moreover, comparison with published stem cell data bases revealed 22 shared genes. A similar approach was taken by Jones et al. Ectopic expression of Upd in drosophila results in enrichment of early germ cells in the testis of these animals. The group then analyzed those testes by gene array to identify genes required for regulation of male germline stem cells. Dr Rosen's group reported molecular characterization of mammary gland stem cells. Gene array analysis revealed 54 unique mammary gland stem cell genes, including members of the Wnt, TNF and MAPK pathways. But importantly, and not surprisingly, when analyzing human ES cells by gene arrays over several passages in culture, Dr Trounson's group found that the cells adapted to the culture conditions, and moreover displayed differences between the individual cell lines. Under the hypothesis that gene expression programs are needed for specific stages of cellular development would be accessible within the chromatin, whereas not required genes would be silenced and deeply embedded in the inactive chromatin (heterochromatin), Azura et al. measured DNA replication timing of key genes. By comparing pluripotent (ES cells) to multipotent (HSC) to unipotent (lymphocytes) cells, the group observed an increase in proportion of late-replicating loci, as cells progress from multipotency to unipotency, and start silencing gene programs that are not needed anymore. It will now be interesting to reconcile these findings with the distinct microRNA expression pattern of stem cells previously reported, and which was correlated by a presentation by Su et al., and the role of certain microRNAs in heterochromatin generation and gene silencing. More specifically, novel factors have been described. Xu et al. described Octap, a novel Oct-4-associtaed protein. Octap interacts specifically with Oct-4 in vitro and in vivo, and when overexpressed in ES cells, reduces Oct-4-driven luciferase reporter activity. Nakano et al. presented that MELK was highly expressed in neurospheres, but not in differentiated cells. MELK (maternal embryonic leucine-zipper kinase), a recently described kinase, had been shown to be expressed in a spatial and temporal pattern during mammalian embryogenesis. By siRNA and overexpression the group now showed a potential role in regulation of neural stem cells.
Epidermal Stem Cells In a transgenic mouse model expressing N-terminally truncated, stabilized b-catenin fused to the ligand-binding domain of a mutant estrogen receptor (DNb-cateninER), under the K14 promoter, Dr Watt's group showed that activation of b-catenin (by topical application of tamoxifen) caused longterm retaining label cells to divide. After 7 days of maintained activation of b-catenin the group observed de novo follicle formation. The extra hair follicle structures formed independently of presence of hair follicle, as they also formed on the foot pad. 21 days of maintained activation resulted in conversion of hair follicles to benign tumors resembling trichofolliculomas, which disappeared after removal of the inducer.
Sharma et al. presented characterization of the luxoid mouse mutant, a mouse with male infertility, which, after birth looses progressively its germline. The group mapped the mutation to the zfp145 gene, a nonsense mutation resulting in a truncated PLZF (Promyelocytic Leukemia Zink Finger) protein. The PLZF protein is a transcriptional repressor that regulates the epigenetic state of the undifferentiated cell and had previously been show to interact with Bmi-1. zfp145-/- mice display the same infertility phenotype as the luxoid mouse, suggesting that PLZF may repress differentiation genes in the germline.
Developmental relationship between HSC and mesenchymal stem cells is still unclear. To investigate the differentiation potency of HSCs into mesenchymal lineage, Matzusaki et al. isolated 'tip'-SP cells with the CD34¯/c-kit+/Sca+/lin¯ surface phenotype from CAG-EGFP transgenic mice and performed single cell transplants into lethally irradiated recipients. After 3 months, the bone marrow of engrafted recipients containing > 60% of donor-derived cells was cultured on fibronectin-coated plates. Very low percentages (0.03%) of the adherent cells were GFP+/CD45¯ and no donor-derived adipocytes or osteocytes could be generated in vitro, indicating that the transplanted cells were already committed to hematopoietic lineage. Neural Progenitor Cells Valponic acid (VPA), a known anti-epileptic and anti-depressant, is an inhibitor of histone deacetylase. Nakashima et al. showed that when culturing adult neural progenitor cells (NPCs) in vitro in absence of serum, but in presence of VPA, the group obtained differentiation into neurons, with a decrease in proliferation. VPA also inhibited astrocyte and oligodendrocyte differentiation. They found reduced histone H3 and H4 acetylation, and increase in NeuroD, SCG10, and synapsin-1 expression. When assayed in vivo, injection of VPA resulted also in an increase in neurons.
Ledermann et al. presented an approach which allowed to obtain ES cell lines from the non-permissible FVB/N mouse strain. The group bred male transgenic mice expressing a tamoxifen-inducible active STAT3 with wt female mice and obtained ES lines from 50-75% of the embryos, when derived in presence of tamoxifen and absence of LIF. Muramatsu et al. developed an efficient method to derive neural stem cells (NSC) from primate ES cells. The NSCs were grafted into the left putamen of a primate model of Parkinson's disease. [11C]L-dopamine uptake was monitored by PET scan and found significantly increased 12 weeks after transplant, and numerous tyrosine hydroxylase-positive neurons were identified by immunohistochemistry at the implantation site. Behavioral recovery was however modest. Majumdar et al. analyzed the immunogenicity of 3 human ES cell lines and found that they did not stimulate allogeneic T cells proliferation in an MLR assay, even after IFN-g stimulation. The ES cells even downregulated T cell responses to third party dendritic cells, in a dose-dependent manner. Neural progenitors were then derived, which like the original ES line did express MHCI and no MHCII. The neuronal progenitors also failed to stimulate T cells in direct MLR assay.
Dr Jaenisch's group used SCNT to distinguish between epigenetic and genetic changes of the genome. The group investigated whether the choice of olfactory receptor, which is different on each individual olfactory neuron, was epigenetic or involved stable gene rearrangement. For this, the group isolated marked individual mature olfactory neurons and used their nuclei to clone mice. Would the receptor choice be genetic, as the T or B cell receptor choice, every single cell in the mice would express the marker. This was however not the case, the group found only a few cells within the olfactory bulb that did express the marker, indicating that the receptor choice is epigenetic.
Recent reports in the drosophila testes and ovarioles have documented the role of the stem cell niche in maintaining the function of stem cells, i.e. self-renewal while also producing progeny. Less is known about the HSC niche. Recent work had shown that osteoblastic cells within the bone marrow regulate stem cell function, through Notch activation. Dr Li's group now presented a role for Bmp1Ra within the hematopoietic stem cell niche and identified discreet areas of HSC activity within the trabecular bone. Conditional cre/lox knock-out mice of Bmp1Ra display an expansion of stem cells in the hair follicle, the intestine and the bone marrow. The mice have ectopic trabecular bone formation, which correlated with a linear increase in HSCs. Chase experiments with BrdU revealed label retaining cells within the bone marrow that were lin+, as well as lin¯ label retaining cells that were attached to the bone surface. The latter population was found increased in the mutant mice. The mutant mice also displayed an increase in N-cadherin+ osteoblastic cells on the bone surface.
Updated: March 18, 2004 |
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