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Stem sense: a proposal for the classification of stem cells.

Stem cells, while difficult to define, hold great promise as tools for understanding development and as therapy. However, this difficulty in defining stem cells has led to a multiplicity of stem cells that may or may not be distinct. The lack of common standards or definitions, the absence of a common forum for discussion, and the range in the ability to manipulate his/her favorite system of stem cells has led to further fragmentation of a field bedeviled by controversy. I suggest that stratification and classification of stem cells on the basis of their function, characteristics, and capabilities would be of enormous benefit to the community. This absence of uniform nomenclature and classification has led to many contradictory claims as to the abilities of stem cells and has made it very difficult to generalize across systems and cell types. I illustrate the problem by providing two examples of how the lack of uniform definitions has slowed progress. I suggest that the effort to establish a consensus on what constitutes a tissue-specific stem cell (definition) and how one would stratify cells (classification) would greatly facilitate progress and perhaps help resolve some of the outstanding controversies.

Animals↗

Haematopoietic stem cells and mesenchymal stem cells as tools for present and future cellular therapies.

Postnatal stem cells are present in many adult tissues, and are thought to ensure homoeostasis by replacing functionally declining cells by newly differentiated ones. Postnatal stem cells used as such or after in vitro manipulation hold out strong hopes for reconstructive therapies. For instance, the grafting of native haematopoietic stem cells (HSC) restores haematopoiesis in genetically deficient individuals or in lethally conditioned leukaemic patients, and systemic injection of in vitro amplified mesenchymal stem cells (MSC) induces recovery of bone growth in patients with osteogenesis imperfecta. Moreover, cells differentiated in vitro from postnatal stem cells exhibiting a specific function can also be used for cell therapy. Myeloid dendritic cells (DC) derived from cultures of HSC may induce tumour-specific cytotoxic T lymphocytes to eradicate the tumour via antigen recognition. In addition, long-lived MSC has been engineered to secrete specific proteins coded by a transgene and used as a source of therapeutic molecules in vivo. All these approaches require large quantities of cells that cannot be obtained (with the exception of HSC) directly from the donor. In vitro procedures allowing the production of therapeutic cells from postnatal stem cells are needed and are at present under development. Below we discuss the rationale and methods currently available for generation of therapeutic cells derived from haematopoietic and mesenchymal stem cells.

Animals↗

The skeletal muscle satellite cell: stem cell or son of stem cell?

The concept of the adult tissue stem cell is fundamental to models of persistent renewal in functionally post-mitotic tissues. Although relatively ignored by stem cell biology, skeletal muscle is a prime example of an adult tissue that can generate terminally differentiated cells uniquely specialized to carry out tissue-specific functions. This capacity is attributed to satellite cells, a population of undifferentiated, quiescent precursors that become activated to divide and differentiate in response to the demands of growth or damage. The aim of this review is to discuss the role of the satellite cell as an adult tissue-specific stem cell. We examine evidence for the presence of behaviourally and phenotypically distinct subpopulations of precursor within the satellite cell pool. Further, we speculate on the possible identity, origins and relevance of multipotent muscle stem cells, a population with both myogenic and hematopoietic potentials that has been isolated from whole muscle. Taken together, current evidence suggests the possibility that the regenerative compartment of adult skeletal muscle may conform to an archetypal stem cell-based hierarchy, maintained within a stem cell niche. It therefore remains to be seen whether all satellite cells are skeletal muscle-specific stem cells, or whether some or all are the progeny of an as yet unidentified muscle stem cell.

Animals↗

Stem cells: the intestinal stem cell as a paradigm.

Stem cell research provides a foundation for therapeutic advancement in oncology, clinical genetics and a diverse array of degenerative disorders. For example, the elucidation of pathways governing proliferative regulation and differentiation within cellular systems will result in medical strategies aimed at the root cause of cancer. At present the characterization of reliable stem cell markers is the immediate aim in this particular field. Over the past 30 years investigators have determined many of the physical and functional properties of stem cells through careful and imaginative experimentation. Intestinal stem cells reside at the crypt base and give rise to all cell types found within the crypt. They readily undergo altruistic apoptosis in response to toxic stimuli although their progeny are hardier and will regain stem cell function to repopulate the tissue compartment, giving rise to the concept of a proliferative hierarchy. Contention exists when deciding whether the full complement of cells within a crypt is derived from either a single or multiple stems. Evidence has also arisen to challenge the long held view that colorectal tumours arise from a single mutated stem cell, as early adenomas from a human XO/XY mosaic contained distinct clones. Mechanisms governing the stem cell cycle and subsequent proliferative activity largely remain obscure. The adenomatous polyposis coli gene product has, however, been shown to promote the degradation of beta-catenin, an enhancer of cell proliferation, thereby downregulating this activity in healthy individuals.

Adenomatous Polyposis Coli↗

Oct4 expression in adult human stem cells: evidence in support of the stem cell theory of carcinogenesis.

The Oct3/4 gene, a POU family transcription factor, has been noted as being specifically expressed in embryonic stem cells and in tumor cells but not in cells of differentiated tissues. With the ability to isolate adult human stem cells it became possible to test for the expression of Oct3/4 gene in adult stem cells and to test the stem cell theory of carcinogenesis. Using antibodies and PCR primers we tested human breast, liver, pancreas, kidney, mesenchyme and gastric stem cells, the cancer cell lines HeLa and MCF-7 and human, dog and rat tumors for Oct4 expression. The results indicate that adult human stem cells, immortalized non-tumorigenic cells and tumor cells and cell lines, but not differentiated cells, express Oct4. Oct4 is expressed in a few cells found in the basal layer of human skin epidermis. The data demonstrate that adult stem cells maintain expression of Oct4, consistent with the stem cell hypothesis of carcinogenesis.

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Relationship between maize stem structural characteristics and resistance to pink stem borer (Lepidoptera: Noctuidae) attack.

The pink stem borer, Sesamia nonagrioides (Lefebvre), is one of the most important insect pests of maize (Zea mays L.) in northwestern Spain. The objectives of this work were to evaluate, at different times during the growth of maize, structural traits related to the entry point and tissues on which larvae feed and to determine the relationship between these structural traits and the stem borer resistance. Six inbred lines with different levels of stem resistance to S. nonagrioides were evaluated in several trials. Potential structural resistance factors included rind and pith puncture resistance (RPR and PPR), rind thickness, length of the meristematic area (LMA), and pith parenchyma interlumen thickness (PPIT). Surprisingly, the inbred lines that showed the strongest stalks, EP42 and EP47, were not stem resistant to pink stem borer attack, while the stem resistant inbreds A509, CM151, and PB130 were among the least resistant to rind puncture. There were no significant differences among resistant and susceptible inbreds for the rind thickness. However, the susceptible inbred EP42 had the softest internode pith, and the resistant inbred PB130 showed the hardest, as was expected. Susceptible inbred lines in general showed higher values for the LMA, while the PPIT was important for individual inbreds. The results suggest that the usefulness of these characters as estimators of pink stem borer resistance is limited to some genotypes. Besides, even among those genotypes, other mechanisms of resistance that do not involve stalk strength could be present. Among the traits considered, the LMA was the most promising as an indicator of resistance to pink stem borer, although further experimentation is necessary.

Animals↗

Neural differentiation of embryonic stem cells induced by conditioned medium from neural stem cell.

Embryonic stem cells can proliferate indefinitely and are capable of differentiating into derivatives of all three embryonic germ layers in vitro, including the neural lineage. The main objective of this study is to test the effects of neural stem cell conditioned medium on the neural differentiation of mouse embryonic stem cells. When cultured in neural stem cell conditioned medium, mouse embryonic stem cells can form floating cell spheres composed of many nestin-positive cells. After trypsinization and growth on gelatin, these embryonic stem cell-derived neural progenitor cells can be expanded for more than 3 months without loss of neural progenitor characteristics. Both neuronal and glial cells can be readily generated from these cells under differentiation conditions. Thus, neural stem cell conditioned medium is a highly potent reagent for inducing the development of mouse embryonic stem cells into the neural lineage, especially neural progenitor cells.

Animals↗

Response to stem bending in forest shrubs: stem or shoot reorientation and shoot release.

Shrubs in the forest understory may be bent by their own weight or by overstory debris. To maintain height growth they must respond to bending by vertical growth of new shoots, reorientation of older axes, or by releasing preventitious buds to form epicormic shoots. I tested for these responses in Ilex verticillata L., Cornus amomum Mill., Gaylussacia baccata (Wang.) K. Koch, Viburnum cassinoides L., Hamamelis virginiana L., and Kalmia latifolia L. For each species, I removed potentially supporting vegetation adjacent to 20 stems, left 10 stems untreated to test for bending by self weight, and bent the remaining 10 stems to 45 degrees to simulate effects of fallen debris. Stem angles and curvatures were measured from before leaf out until just before leaf fall to detect either sagging from self weight or upward bending from tension wood action. Control stems initially leaned out of vertical and five of six species sagged further into a cantilever form. Several control stems failed and bent to the ground. Stems of H. virginiana, I. verticillata, and C. amomum formed tension wood, but only the first two species bent upward. Viburnum cassinoides, G. baccata, and K. latifolia formed no tension wood and sagged further down after being bent. Epicormic shoots formed with varying frequencies in all species except K. latifolia. Epicormic shoots were the major response in C. amomum, V. cassinoides, and G. baccata. New terminal shoots on bent stems recovered toward vertical in I. verticillata and K. latifolia. Negative gravitropic response of shoots was the only recovery mechanism for K. latifolia.

Forestry↗

Kinetics and regulation of human keratinocyte stem cell growth in short-term primary ex vivo culture. Cooperative growth factors from psoriatic lesional T lymphocytes stimulate proliferation among psoriatic uninvolved, but not normal, stem keratinocytes.

Flow cytometric analysis of primary ex vivo keratinocyte cultures demonstrated that stem cells, (beta 1 integrin+, keratin 1/keratin 10 [K1/K10-], proliferating cell nuclear antigen [PCNA-] [Bata-Csorgo, Zs., C. Hammerberg, J. J. Voorhees, and K. D. Cooper. 1993. J. Exp. Med. 178:1271-1281]) establish such cultures. This methodology also enabled the quantitation of synchronized recruitment of these cells from G0 into G1 of the cell cycle (PCNA expression), which preceded bright beta 1 integrin expression. (beta 1 integrinbright expression has been shown to be a characteristic feature of keratinocyte stem cells in culture (Jones, P. H., and F. M. Watt. 1993. Cell. 73:713-724). Using the above assay, we determined whether lesional T lymphocytes in psoriasis could be directly responsible for the induction of the stem cell hyperproliferation that is characteristic of this disease. Indeed, CD4+ T lymphocytes, cloned from lesional psoriatic skin and stimulated by immobilized anti-CD3 plus fibronectin, promoted psoriatic uninvolved keratinocyte stem cell proliferation via soluble factors. This induction appeared to be through accelerated recruitment of stem cells from their quiescent state (G0) into cell cycle. By contrast, normal keratinocyte stem cells exhibited no such growth stimulation. Supernatants exhibiting growth induction all contained high levels of GM-CSF and gamma-IFN, low IL-3 and TNF-alpha, and variable IL-4. Only anti-gamma-IFN antibody was able to neutralize growth stimulatory activity of the supernatants on psoriatic uninvolved keratinocyte stem cells. However, because recombinant gamma-IFN alone inhibited growth in this assay, these data suggest that, in psoriasis, gamma-IFN acts cooperatively with other growth factors in the immune induction of cell cycle progression by the normally quiescent stem cell keratinocytes.

CD4 Antigens↗

Comments about possible use of human embryonic stem cell-derived cardiomyocytes to direct autologous adult stem cells into the cardiomyogenic lineage.

Several studies have shown that cell-transplantation therapy following myocardial infarction has some efficacy in aiding myocardial repair and subsequent recovery of heart function. Large-scale production of human embryonic stem cell-derived cardiomyocytes can potentially provide an abundant supply of donor cells for myocardial transplantation. There are, however, immunological barriers to their use in human clinical therapy.A novel approach would be to look at utilizing human embryonic stem cell-derived cardiomyocytes to reprogram autologous adult stem cells to express cardiomyogenic function, instead of using these directly for transplantation. This could be achieved through a number of novel techniques. Enucleated cytoplasts generated from human embryonic stem cell-derived cardiomyocytes could be fused with autologous adult stem cells to generate cytoplasmic hybrids or cybrids. Adult stem cells could also be temporarily permeabilized and exposed to cytoplasmic extracts derived from these cardiomyocytes. Alternatively, intact cells or enucleated cytoplasts from human embryonic stem cell-derived cardiomyocytes could be co-cultured with adult stem cells in vitro, to provide the cellular contacts and electrical coupling that might enable some degree of trans-differentiation to take place. This review would therefore examine the potential advantages and disadvantages of such a novel approach, in comparison to other more conventional techniques such as the use of exogenous cytokines/growth factors or the use of genetic modulation.

Adult↗

Hematopoietic stem cells: the paradigmatic tissue-specific stem cell.

The recent prospective isolation of a wide variety of somatically derived stem cells has affirmed the notion that homeostatic maintenance of most tissues and organs is mediated by tissue-specific stem and progenitor cells and fueled enthusiasm for the use of such cells in strategies aimed at repairing or replacing damaged, diseased, or genetically deficient tissues and organs. Hematopoietic stem cells (HSCs) are arguably the most well-characterized tissue-specific stem cell, with decades of basic research and clinical application providing not only a profound understanding of the principles of stem cell biology, but also of its potential pitfalls. It is our belief that emerging stem cell fields can benefit greatly from an understanding of the lessons learned from the study of HSCs. In this review we discuss some general concepts regarding stem cell biology learned from the study of HSCs with a highlight on recent work pertaining to emerging topics of interest for stem cell biology.

Animals↗

[Therapeutic use of stem cells. II. Adult stem cells].

Many degenerative diseases are not curable by means of classical medicine. The long term objective of cell therapy is to treat the patients with their own stem cells that could be either purified from the diseased organ or from "reservoirs" of stem cells such as that constituted by the bone marrow. The existence of stem cells in the organs or reservoirs is now established in vitro and in some cases, in animal models. Numbers of technical problems linked to the scarcity of these cells still delay the clinical use of purified stem cells. However, clinical protocols using heterogeneous cell populations have already started to treat a growing number of diseases. In some case, autologous cells can be used, as it is the case for bone marrow transplantation in blood diseases. Mesenchymal cells, also purified from the bone marrow are currently used in orthopaedic diseases. Because these cells reveal a broad differentiation potential, active research programs explore their possible use for treatment of other diseases. Bone marrow also contains vascular stem cells that could be active in reappearing defective vessels responsible for ischaemic diseases. Indeed, clinical trials in which bone marrow cells are injected in the cardiac muscle of patients with myocardial infarction or in the leg muscle (gastrocnemius) of patients with hind limb ischaemia have already started. Artificial skin prepared from skin biopsies is used for the reconstitution of the derma of severely burned patients. Clinical trials have also started, using allogenic cells. The patients must be treated by immunosuppressive drugs. Neurodegenerative diseases such as Parkinson have been successfully treated by intra-cerebral injection of foetal neurones. Pancreatic islets implanted in the liver have shown to re-establish a normal glycaemia in diabetic patients. However, all these clinical trials use differentiated cells or at least progenitors which display differentiation potential and lifetime much more restricted than those of stem cell. Numbers of laboratories are currently working to improve stem cell purification and expansion. This is a prerequisite to use these stem cells as a more efficient second generation cell therapy product.

Adult↗

Proliferation of purified murine hemopoietic stem cells in serum-free cultures stimulated with purified stem-cell-activating factor.

Under conditions of steady-state hemopoiesis in normal mice, the majority of hemopoietic stem cells in the bone marrow are in the quiescent state of the cell cycle. These cells can be stimulated to proliferate in vitro by the addition of a factor termed the "stem-cell-activating factor" (SAF), which is present in medium conditioned by various cell types. This factor is indistinguishable in antigenic and molecular properties from the lymphokine interleukin 3 (IL-3). The action of SAF on the stem cell cycle was studied by examination of the survival of the spleen colony-forming unit(s) (CFU-S) after four days of serum-free culture in the presence of purified SAF. CFU-S subtypes were distinguished on the basis of the day of colony counting (i.e., days 7, 9, and 12 after transplantation). The results indicate that SAF selectively induces an increase of the day-7 CFU-S: the CFU-S number increased 2.7-fold on day 7 and 1.2-fold on day 9, and decreased fivefold for day-12 CFU-S. Similar results were obtained in SAF culture of partially and highly purified stem cells. The proliferation of day-9 CFU-S in cultures of low-density bone marrow cells was found to be similar to that of unfractionated bone marrow cells until day 4 of culture. However, in the culture of partially purified stem cells, this proliferation stopped between days 4 and 10, whereas it continued with unfractionated cells. This indicates that cocultured bone marrow cells affect the proliferation of stem cells upon induction by SAF; day-4 cultures of highly purified resting stem cells with purified SAF resulted in a similar decrease in day-12 CFU-S and an increase in day-7 CFU-S, as was observed with unfractionated bone marrow cells. The DNA histogram of the stimulated sorted cells clearly revealed an actively DNA-synthesizing population. The results are in agreement with those of a selective induction of proliferation by SAF of resting pluripotent hemopoietic stem cells.

Animals↗

Alternative sources of adult stem cells: a possible solution to the embryonic stem cell debate.

The complex moral and ethical debate surrounding the definition of the origins of human life, together with conflicting current and proposed legislation on state and federal levels, is hindering the course of research into the therapeutic uses of human embryonic stem cells. However, newly identified sources of adult stem cells, free from many of the ethical and legal concerns attached to embryonic stem cell research, may offer great promise for the advancement of medicine. These alternative sources may alleviate the need to resolve the stem cell debate before further therapeutic benefits of stem cell research can be realized. While legislation and ethics evolve to address the legal and moral issues of embryonic stem cell research, innovative researchers will continue to search for and find real and present solutions for cell-based therapies using adult stem cells.

Adult Stem Cells↗

Signaling pathways in self-renewing hematopoietic and leukemic stem cells: do all stem cells need a niche?

Many adult tissue stem cells, such as the cells of the hematopoietic system, gastrointestinal epithelium, brain, epidermis, mammary gland and lung have now been identified, all of them fulfilling a crucial role in supplying organisms with mature cells during normal homeostasis as well as in times of tissue generation or repair. Two unique features characterize adult stem cells: the ability to generate new pluripotent stem cells (to self-renew) and the ability to give rise to differentiated progeny that has lost its self-renewal capacity. Our understanding of the mechanisms that determine whether, where and when a stem cell will self-renew or differentiate is still limited, but recent advances have indicated that the stem cell microenvironment, or niche, provides essential cues that direct these cell fate decisions. Moreover, loss of control over these cell fate decisions might lead to cellular transformation and cancer. This review addresses the current understandings of the molecular mechanisms that regulate hematopoietic stem cell self-renewal in the niche and how leukemic transformation might change the dependency of leukemic stem cells on their microenvironment for self-renewal and survival.

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Pilot trial of interleukin-2 with granulocyte colony-stimulating factor for the mobilization of progenitor cells in advanced breast cancer patients undergoing high-dose chemotherapy: expansion of immune effectors within the stem-cell graft and post-stem-cell infusion.

PURPOSE: To evaluate whether administration of interleukin-2 (IL-2) with granulocyte colony-stimulating factor (G-CSF) improves mobilization of immune effector cells into the stem-cell graft of patients undergoing high-dose chemotherapy and autografting. PATIENTS AND METHODS: We performed a trial of stem-cell mobilization with IL-2 and G-CSF in advanced breast cancer patients receiving high-dose chemotherapy with cyclophosphamide, thiotepa, and carboplatin and stem cells followed by IL-2. The trial defined immune, hematologic, and clinical effects of IL-2 in this setting. RESULTS: Of 32 patients enrolled, nine received G-CSF alone for mobilization. Twenty-one of 23 patients mobilized with IL-2 plus G-CSF had stem cells collected with more mononuclear cells than those receiving G-CSF (19.3 v 10.4 x 10(8)/kg; P =.006), but fewer CD34(+) progenitor cells (6.9 v 22.0 x 10(6)/kg; P =.049). The IL-2 plus G-CSF-mobilized patients had greater numbers of activated T (CD3(+)/CD25(+)) cells (P =.009), natural killer (NK; CD56(+)) cells (P =.007), and activated NK (CD56 bright(+)) cells (P: =.039) than those patients mobilized with G-CSF. NK (P =.042) and lymphokine-activated killer (LAK) (P =.016) activity was increased in those mobilized with IL-2 + G-CSF, whereas G-CSF-mobilized patients had a decline in cytolytic activity. In the third week posttransplantation, immune reconstitution was superior in those mobilized with IL-2 plus G-CSF based on greater numbers of activated T cells (P =.003), activated NK cells (P =.04), and greater LAK activity (P =.003). The 16 of 21 IL-2 + G-CSF-mobilized patients with adequate numbers of stem cells (> 1.5 x 10(6) CD34(+) cells/kg) collected engrafted rapidly posttransplantation. CONCLUSION: The results demonstrate that G-CSF + IL-2 can enhance the number and function of antitumor effector cells in a mobilized autograft without impairing the hematologic engraftment, provided that CD34 cell counts are more than 1.5 x 10(6) cells/kg. Mobilization of CD34(+) stem cells does seem to be adversely affected. In those mobilized with IL-2 and G-CSF, post-stem-cell immune reconstitution of antitumor immune effector cells was enhanced.

Adult↗

Current basic research of hematopoietic stem cells in China and comments on stem cell plasticity.

The basic studies selected were mainly published since 1998 and related to stem cell biology and engineering and particularly the efforts for developing new sources of hematopoietic stem/progenitor cells ex vivo. Hematopoietic cells and lymphocytes can be developed by induced differentiation in a appropriate way of culture, originating in the embryo- or adult-derived stem cells or tissue-committed stem cells which still exist in the tissue of adults. The most primitive multipotential embryonic stem cell from embryo or adult tissue has the plasticity to differentiate into every kind of progenies, the committed tissue-specific stem cell, by different proper ways of induction in vitro. The committed tissue-specific stem cell, however, can only be induced to differentiate along the line of its committed origin of tissue. No studies in China strongly confirmed yet the existence of "transdifferentiation" among the tissue- or organ-specific stem cells.

Adult↗

Circulating primitive stem cells in paroxysmal nocturnal hemoglobinuria (PNH) are predominantly normal in phenotype but granulocyte colony-stimulating factor treatment mobilizes mainly PNH stem cells.

Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired hemolytic anemia resulting from a somatic mutation in a hemopoietic stem cell. In most cases of hemolytic PNH, the majority of the marrow cells are derived from the PNH clone. Recent evidence has indicated, however, that the majority of the most primitive peripheral blood stem cells (PBSCs) in PNH appear to be of normal phenotype. This has led to tentative suggestions that normal PBSCs could be collected and used for autologous transplantation. We have investigated this possibility in four PNH patients by treating them with granulocyte colony-stimulating factor (G-CSF) in an attempt to mobilize normal progenitors. The expression of glycosylphosphatidylinositol (GPI)-linked proteins was analyzed by flow cytometry on mature neutrophils, late stem cells (CD34+/CD38+), and primitive stem cells (CD34+/CD38-). The phenotyping and stem cell quantitation was performed in steady-state blood and post-G-CSF administration. The most primitive PBSCs (CD34+/CD38-) were almost all normal before G-CSF treatment, even when the patients' neutrophils were mainly PNH. However, after G-CSF, the cells that were mobilized into the peripheral blood were of a similar phenotype to the mature neutrophils, ie, mainly PNH. It is possible that PNH-stem cells are preferentially destroyed by complement in the peripheral blood leaving only normal cells in the circulation. After G-CSF, the PNH cells in the marrow are released into the blood. Our findings suggest that it would be difficult to collect sufficient numbers of normal stem cells for autologous transplantation.

Adult↗