[Stem cells in adults]. / Stamceller hos voksne.

BACKGROUND: We present a literature review of the plasticity observed by adult stem cells. MATERIALS AND METHODS: We have reviewed the literature regarding stem cells from adults in order to summarise their ability to generate cells of other types than those of the tissue/organ from which they were isolated. RESULTS: Adult stem cells have recently been demonstrated to terminally differentiate into cells of other tissues than those from which they were originally isolated. For example, bone marrow cells have been shown to generate liver, nerve, heart and skeletal muscle cells in addition to their well-known ability to produce blood and mesenchymal cells. INTERPRETATION: Most studies demonstrate a proof-of-principle in animal models; much more research is needed before adult stem cells can be utilised in human medicine. However, the published reports are encouraging and give reasons for a cautious optimism with regard to future clinical use.

Self-renewal of multipotent long-term repopulating hematopoietic stem cells is negatively regulated by Fas and tumor necrosis factor receptor activation.

Multipotent self-renewing hematopoietic stem cells (HSCs) are responsible for reconstitution of all blood cell lineages. Whereas growth stimulatory cytokines have been demonstrated to promote HSC self-renewal, the potential role of negative regulators remains elusive. Receptors for tumor necrosis factor (TNF) and Fas ligand have been implicated as regulators of steady-state hematopoiesis, and if overexpressed mediate bone marrow failure. However, it has been proposed that hematopoietic progenitors rather than stem cells might be targeted by Fas activation. Here, murine Lin(-)Sca1(+)c-kit(+) stem cells revealed little or no constitutive expression of Fas and failed to respond to an agonistic anti-Fas antibody. However, if induced to undergo self-renewal in the presence of TNF-alpha, the entire short and long-term repopulating HSC pool acquired Fas expression at high levels and concomitant activation of Fas suppressed in vitro growth of Lin(-)Sca1(+)c-kit(+) cells cultured at the single cell level. Moreover, Lin(-)Sca1(+)c-kit(+) stem cells undergoing self-renewal divisions in vitro were severely and irreversibly compromised in their short- and long-term multilineage reconstituting ability if activated by TNF-alpha or through Fas, providing the first evidence for negative regulators of HSC self-renewal.

Upregulation of Flt3 expression within the bone marrow Lin(-)Sca1(+)c-kit(+) stem cell compartment is accompanied by loss of self-renewal capacity.

Flt3 has emerged as a potential regulator of hematopoietic stem cells (HSC). Sixty percent of cells in the mouse marrow Lin(-)Sca1(+)c-kit(+) HSC pool expressed flt3. Although single cell cloning showed comparable high proliferative, myeloid, B, and T cell potentials of Lin(-)Sca1(+)c-kit(+)flt3(+) and Lin(-)Sca1(+)c-kit(+)flt3(-) cells, only Lin(-)Sca1(+)c-kit(+)flt3(-) cells supported sustained multilineage reconstitution. In striking contrast, Lin(-)Sca1(+)c-kit(+)flt3(+) cells rapidly and efficiently reconstituted B and T lymphopoiesis, whereas myeloid reconstitution was exclusively short term. Unlike c-kit, activation of flt3 failed to support survival of HSC, whereas only flt3 mediated survival of Lin(-)Sca1(+)c-kit(+)flt3(+) reconstituting cells. Phenotypic and functional analysis support that Lin(-)Sca1(+)c-kit(+)flt3(+) cells are progenitors for the common lymphoid progenitor. Thus, upregulation of flt3 expression on Lin(-)Sca1(+)c-kit(+) HSC cells is accompanied by loss of self-renewal capacity but sustained lymphoid-restricted reconstitution potential.

[Intracytoplasmic sperm injection--risk of abnormalities]. / Intracytoplasmatisk spermieinjeksjon--risiko for misdannelser.

BACKGROUND: Intracytoplasmic sperm injection (ICSI) is performed in a large number of fertility clinics; since 1991, several thousand children have been conceived by this method. Intracytoplasmic sperm injection is different than traditional in vitro-fertilization (IVF) in that a single spermatozoon is mechanically transferred into a mature oocyte using a glass pipette. Concern has been raised as to whether intracytoplasmic sperm injection damages the chromosomes and/or the spindle apparatus in the oocyte, leading to an increased risk of congenital abnormalities. MATERIAL AND METHODS: We have reviewed the literature on congenital abnormalities in children conceived by intracytoplasmatic sperm injection. RESULTS AND INTERPRETATION: Although the reports are methodologically heterogeneous, most articles conclude that intracytoplasmatic sperm injection does not cause a statistically significant increased risk of congenital abnormalities compared to traditional in vitro fertilization or normally conceived children. However, some studies do report an increased risk. The issue cannot be finally settled until even larger studies on children conceived by intracytoplasmatic sperm injection are published.

Distinct requirements for optimal growth and In vitro expansion of human CD34(+)CD38(-) bone marrow long-term culture-initiating cells (LTC-IC), extended LTC-IC, and murine in vivo long-term reconstituting stem cells.

Recently, primitive human bone marrow (BM) progenitors supporting hematopoiesis in extended (>60 days) long-term BM cultures were identified. Such extended long-term culture-initiating cells (ELTC-IC) are of the CD34(+)CD38(-) phenotype, are quiescent, and are difficult to recruit into proliferation, implicating ELTC-IC as the most primitive human progenitor cells detectable in vitro. However, it remains to be established whether ELTC-IC can proliferate and potentially expand in response to early acting cytokines. Here, CD34(+)CD38(-) BM ELTC-IC (12-week) were efficiently recruited into proliferation and expanded in vitro in response to early acting cytokines, but conditions for expansion of ELTC-IC activity were distinct from those of traditional (5-week) LTC-IC and murine long-term repopulating cells. Whereas c-kit ligand (KL), interleukin-3 (IL-3), and IL-6 promoted proliferation and maintenance or expansion of murine long-term reconstituting activity and human LTC-IC, they dramatically depleted ELTC-IC activity. In contrast, KL, flt3 ligand (FL), and megakaryocyte growth and development factor (MGDF) (and KL + FL + IL-3) expanded murine long-term reconstituting activity as well as human LTC-IC and ELTC-IC. Expansion of LTC-IC was most optimal after 7 days of culture, whereas optimal expansion of ELTC-IC activity required 12 days, most likely reflecting the delayed recruitment of quiescent CD34(+)CD38(-) progenitors. The need for high concentrations of KL, FL, and MGDF (250 ng/mL each) and serum-free conditions was more critical for expansion of ELTC-IC than of LTC-IC. The distinct requirements for expansion of ELTC-IC activity when compared with traditional LTC-IC suggest that the ELTC-IC could prove more reliable as a predictor for true human stem cell activity after in vitro stem cell manipulation.

Lymphoid-restricted development from multipotent candidate murine stem cells: distinct and complimentary functions of the c-kit and flt3-ligands.

The two tyrosine kinase receptors, c-kit and flt3, and their respective ligands KL and FL, have been demonstrated to play key and nonredundant roles in regulating the earliest events in hematopoiesis. However, their precise roles and potential interactions in promoting early lymphoid commitment and development remain unclear. Here we show that most if not all murine Lin(-/lo)Sca1(+)c-kit(+) bone marrow (BM) cells generating B220(+)CD19(+) proB-cells in response to FL and interleukin-7 (IL-7) also have a myeloid potential. In contrast to FL + IL-7, KL + IL-7 could not promote proB-cell formation from Lin(-/lo)Sca1(+)c-kit(+) cells. However, KL potently enhanced FL + IL-7-stimulated proB-cell formation, in part through enhanced recruitment of FL + IL-7-unresponsive Lin(-/lo)Sca1(+)c-kit(+) progenitors, and in part by enhancing the growth of proB-cells. The enhanced recruitment (4-fold) in response to KL occurred exclusively from the Lin(-/lo)Sca1(+)c-kit(+)flt3(-) long-term repopulating stem cell population, whereas KL had no effect on FL + IL-7-stimulated recruitment of Lin(-/lo)Sca1(+)c-kit(+)flt3(+) short-term repopulating cells. The progeny of FL + IL-7-stimulated Lin(-/lo)Sca1(+)c-kit(+) cells lacked in vitro and in vivo myeloid potential, but efficiently reconstituted both B and T lymphopoiesis. In agreement with this FL, but not KL, efficiently induced expression of B220 and IL-7 receptor-alpha on Lin(-/lo)Sca1(+)c-kit(+)flt3(+) cells. Thus, whereas KL appears crucial for recruitment of FL + IL-7-unresponsive candidate (c-kit(+)flt3(-)) murine stem cells, FL is essential and sufficient for development toward lymphoid restricted progenitors from a population of (c-kit(+)flt3(+)) multipotent short-term reconstituting progenitors.

Bidirectional effect of interleukin-10 on early murine B-cell development: stimulation of flt3-ligand plus interleukin-7-dependent generation of CD19(-) ProB cells from uncommitted bone marrow progenitor cells and growth inhibition of CD19(+) ProB cells.

B-cell commitment and early development from multipotent hematopoietic progenitor cells has until recently been considered to be dependent on direct interaction with stromal cells. We recently showed that the flt3 ligand (FL) has a unique ability to interact with interleukin-7 (IL-7) to directly and selectively promote B-cell development from murine bone marrow progenitor cells with a combined myeloid and lymphoid potential. Here we report that whereas IL-10 alone has no ability to stimulate growth of primitive (Lin-Sca-1(+)c-kit+) bone marrow progenitor cells, it potently enhances FL + IL-7-induced proliferation (sevenfold). This enhanced proliferation results from recruitment of progenitors unresponsive to FL + IL-7 alone, as well as from increased growth of individual clones, resulting in a 7,000-fold cellular expansion over 12 days. Single cell cultures and delayed addition studies suggested that the stimulatory effect of IL-10 was directly mediated on the progenitor cells. The cells generated in response to FL + IL-7 + IL-10 appeared to be almost exclusively proB cells, as shown by their expression of B220, CD24, CD43, and lack of expression of c mu, myeloid, erythroid, and T-cell surface antigens. Although IL-10 also enhanced kit ligand (KL) + IL-7-induced proliferation of Lin-Sca-1(+)c-kit+ progenitor cells, the resulting cells were predominantly myeloid progeny. Accordingly, FL + IL-7 + IL-10 was 100-fold more efficient in stimulating production of proB cells than KL + IL-7 + IL-10. In contrast to its ability to stimulate the earliest phase of proB cell formation and proliferation, IL-10 inhibited growth of proB cells generated in response to FL + IL-7. Analysis of CD19 expression on cells generated in FL + IL-7 + IL-10 showed that almost all cells generated under these conditions lacked expression of CD19, in contrast to cells generated in the absence of IL-10, which were predominantly CD19(+). Replating of sorted CD19(+) and CD19(-) proB cells in FL + IL-7 or FL + IL-7 + IL-10 showed that IL-10 efficiently blocked growth of CD19(+), but not CD19(-) cells. Both CD19(-) and CD19(+) cells expressed lambda5 and VpreB , shown to be specific for B-cell progenitors. In addition, sorted CD19(-) cells generated CD19(+) cells in response to FL + IL-7. Thus, IL-10 has a dual regulatory effect on early B-cell development from primitive murine bone marrow progenitor cells in that it enhances FL + IL-7-induced proB-cell formation and growth before acquisition of CD19 expression, whereas growth of CD19(+) proB cells is inhibited.

Transforming growth factor-beta1 abrogates Fas-induced growth suppression and apoptosis of murine bone marrow progenitor cells.

Fas, a member of the tumor necrosis factor (TNF ) receptor superfamily is a critical downregulator of cellular immune responses. Proinflammatory cytokines like interferon-gamma (IFN-gamma) and TNF-alpha can induce Fas expression and render hematopoietic progenitor cells susceptible to Fas-induced growth suppression and apoptosis. Transforming growth factor-beta1 (TGF-beta1 ) is an essential anti-inflammatory cytokine, thought to play a key role in regulating hematopoiesis. In the present studies we investigated whether TGF-beta1 might regulate growth suppression and apoptosis of murine hematopoietic progenitor cells signaled through Fas. In the presence of TNF, activation of Fas almost completely blocked clonogenic growth of lineage-depleted (Lin-) bone marrow (BM) progenitor cells in response to granulocyte-macrophage colony-stimulating factor (GM-CSF ), CSF-1, or a combination of multiple cytokines. Whereas TGF-beta1 alone had no effect or stimulated growth in response to these cytokines, it abrogated Fas-induced growth suppression. Single-cell studies and delayed addition of TGF-beta1 showed that the ability of TGF-beta1 to inhibit Fas-induced growth suppression was directly mediated on the progenitor cells and not indirect through potentially contaminating accessory cells. Furthermore, TGF-beta1 blocked Fas-induced apoptosis of Lin- BM cells, but did not affect Fas-induced apoptosis of thymocytes. TGF-beta1 also downregulated the expression of Fas on Lin- BM cells. Thus, TGF-beta1 potently and directly inhibits activation-dependent and Fas-mediated growth suppression and apoptosis of murine BM progenitor cells, an effect that appears to be distinct from its ability to induce progenitor cell-cycle arrest. Consequently, TGF-beta1 might act to protect hematopoietic progenitor cells from enhanced Fas expression and function associated with proinflammatory responses.

Ability of early acting cytokines to directly promote survival and suppress apoptosis of human primitive CD34+CD38- bone marrow cells with multilineage potential at the single-cell level: key role of thrombopoietin.

Purified primitive progenitor/stem cells from bone marrow represent likely target populations for ex vivo expansion of stem cells to be used in high-dose chemotherapy or gene therapy. Whereas such primitive progenitor cells require combined stimulation by multiple cytokines for growth, some cytokines selectively promote viability rather than growth when acting individually. We investigated here for the first time the direct effects of cytokines on survival of primitive CD34+CD38- human bone marrow progenitor cells at the single-cell level. Interleukin-3 (IL-3) and the ligands for c-kit (KL) and flt3 (FL) had direct and selective viability-promoting effects on a small fraction of CD34+CD38- but not CD34+CD38+ progenitor cells. Interestingly, the recently cloned thrombopoietin (Tpo), although stimulating little growth, kept most CD34+CD38- progenitors viable after prolonged culture, maintaining twofold and fourfold more progenitors viable than KL and IL-3, respectively. A high fraction of these progenitors had a combined myeloid and erythroid differentiation potential, as well as capacity for prolonged production of progenitor cells under stroma-independent conditions. In addition, Tpo promoted viability of CD34+CD38- long-term culture-initiating cells, further supporting the idea that Tpo promotes viability of primitive human progenitor cells. Finally, Tpo suppressed apoptosis of CD34+CD38- cells in culture. Thus, the present studies show a novel effect of Tpo, implicating a potential role of this cytokine in maintaining quiescent primitive human progenitor cells viable.

Thrombopoietin promotes adhesion of primitive human hemopoietic cells to fibronectin and vascular cell adhesion molecule-1: role of activation of very late antigen (VLA)-4 and VLA-5.

Thrombopoietin (Tpo), the ligand for c-mpl and a principal regulator of megakaryocytopoiesis and platelet production, has been demonstrated to stimulate the growth and differentiation of megakaryocyte as well as multipotent hemopoietic progenitor cells. In the present study we demonstrate that Tpo can stimulate the adhesion of the Mo7e progenitor cell line to fibronectin (Fn) as well as vascular cell adhesion molecule-1 through activation of very late antigen (VLA)-4 and VLA-5, adhesion molecules previously demonstrated to be involved in regulation of steady state hemopoiesis. Tpo-induced adhesion was concentration dependent, reached a maximum following 30 min, and appeared to be dependent on adenylate cyclase, and tyrosine kinase activity. Furthermore, second messenger inhibitors implicated essential and complimentary roles of phosphatidylinositol-3-kinase and protein kinase C in mediating Tpo-induced adhesion. The ability of Tpo to promote adhesion to fibronectin was comparable to that of IL-3, but less than that of stem cell factor. Unlike the ability of these cytokines to synergistically enhance growth of Mo7e as well as normal progenitor cells, no synergy was observed with regard to their ability to enhance adhesion. Finally, Tpo stimulated adhesion of primitive (CD34+ CD38-) human bone marrow cells to fibronectin, predominantly through activation of VLA-5, whereas no such effect could be observed on CD34+ CD38+ bone marrow cells. Thus, Tpo might play an important role in early hemopoiesis, at least in part through its ability to promote adhesion through activation of adhesion molecules on hemopoietic progenitor cells.

Thrombopoietin directly and potently stimulates multilineage growth and progenitor cell expansion from primitive (CD34+ CD38-) human bone marrow progenitor cells: distinct and key interactions with the ligands for c-kit and flt3, and inhibitory effects of TGF-beta and TNF-alpha.

Thrombopoietin (Tpo) is a primary regulator of megakaryocyte and platelet production. However, studies in c-mpl-deficient mice suggest that Tpo might also play an important role in early hemopoiesis. Here, the direct ability of Tpo to stimulate stroma-independent growth, multilineage differentiation, and progenitor cell expansion from single primitive CD34+ CD38- human bone marrow cells was investigated. Tpo alone stimulated limited clonal growth, but synergized with c-kit ligand (KL), flt3 ligand (FL), or IL-3 to potently enhance clonogenic growth. Whereas KL and FL in combination stimulated the clonal growth of only 3% of CD34+ CD38- cells, 40% of CD34+ CD38- cells were recruited by KL+FL+Tpo, demonstrating that Tpo promotes the growth of a high fraction of CD34+ CD38- progenitor cells. Additional cytokines (IL-3, IL-6, and erythropoietin (Epo)) did not significantly enhance clonal growth above that observed in response to KL+FL+Tpo. In contrast, Tpo enhanced clonogenic growth in response to KL+FL+IL-3+IL-6+Epo by as much as 80%, implicating a key role for this cytokine in early hemopoiesis. Importantly, we also demonstrate that the majority of Tpo-recruited CD34+ CD38- progenitor cells have a multilineage differentiation potential, and that Tpo promotes prolonged expansion of multipotent progenitors. Specifically, whereas progenitor cells were reduced in cultures containing only KL+FL, addition of Tpo resulted in 40-fold expansion of multipotent progenitors following a 14-day incubation. Finally, we identified inhibitors of Tpo-induced progenitor cell growth, in that TGF-beta as well as TNF-alpha almost completely abrogated the growth of CD34+ CD38- progenitor cells in response to Tpo alone as well as KL+FL+Tpo.

Thrombopoietin, but not erythropoietin, directly stimulates multilineage growth of primitive murine bone marrow progenitor cells in synergy with early acting cytokines: distinct interactions with the ligands for c-kit and FLT3.

Thrombopoietin (Tpo), the ligand for c-mpl, has been shown to be the principal regulator of megakaryocytopoiesis and platelet production. The ability of Tpo to potently stimulate the growth of committed megakaryocyte (Mk) progenitor cells has been studied in detail. Murine fetal liver cells, highly enriched in primitive progenitors, have been shown to express c-mpl, but little is known about the ability of Tpo to stimulate the growth and differentiation of primitive multipotent bone marrow (BM) progenitor cells. Here, we show that Tpo alone and in combination with early acting cytokines can stimulate the growth and multilineage differentiation of Lin- Sca-1+ BM progenitor cells. In particular, Tpo potently synergized with the ligands for c-kit (stem cell factor [SCF]) and flt3 (FL) to stimulate an increase in the number and size of clones formed from Lin- Sca-1+ progenitors. When cells were plated at 1 cell per well, the synergistic effect of Tpo was observed both in fetal calf serum-supplemented and serum-depleted medium and was decreased if the addition of Tpo to cultures was delayed for as little as 24 hours, suggesting that Tpo is acting directly on the primitive progenitors. Tpo added to SCF + erythropoietin (Epo)-supplemented methylcellulose cultures potently enhanced the formation of multilineage colonies containing granulocytes, macrophages, erythrocytes, and Mks. SCF potently enhanced Tpo-stimulated production of high-ploidy Mks from Lin- Sca-1+ progenitors, whereas the increased growth response obtained when combining Tpo with FL did not translate into increased Mk production. The ability of Tpo and SCF to synergistically enhance the growth of Lin- Sca-1+ progenitors was predominantly observed in the more primitive rhodamine 123(lo) fraction. Tpo also enhanced growth of Lin- Sca-1+ progenitors when combined with interleukin-3 (IL-3) and IL-11 but not with IL-12, granulocyte colony-stimulating factor, granulocyte-macrophage colony-stimulating factor, or Epo. Epo, which has high homology to Tpo, was unable to stimulate the growth of Lin- Sca-1+ progenitors alone or in combination with SCF or FL, suggesting that c-mpl is expressed on more primitive stages of progenitors than the Epo receptor. Thus, the present studies show the potent ability of Tpo to enhance the growth of primitive multipotent murine BM progenitors in combination with multiple early acting cytokines and documents its unique ability to synergize with SCF to enhance Mk production from such progenitors.

Thrombopoietin, but not erythropoietin promotes viability and inhibits apoptosis of multipotent murine hematopoietic progenitor cells in vitro.

The recently cloned c-mpl ligand, thrombopoietin (Tpo), has been extensively characterized with regard to its ability to stimulate the growth, development, and ploidy of megakaryocyte progenitor cells and platelet production in vitro and in vivo. Primitive hematopoietic progenitors have been shown to express c-mpl, the receptor for Tpo. In the present study, we show that Tpo efficiently promotes the viability of a subpopulation of Lin-Sca-1+ bone marrow progenitor cells. The ability of Tpo to maintain viable Lin-Sca-1+ progenitors was comparable to that of granulocyte colony-stimulating factor and interleukin-1, whereas stem cell factor (SCF) promoted the viability of a higher number of Lin-Sca-1+ progenitor cells when incubated for 40 hours. However, after prolonged (> 40 hours) preincubation, the viability-promoting effect of Tpo was similar to that of SCF. An increased number of progenitors surviving in response to Tpo had megakaryocyte potential (37%), although almost all of the progenitors produced other myeloid cell lineages as well, suggesting that Tpo acts to promote the viability of multipotent progenitors. The ability of Tpo to promote viability of Lin-Sca-1+ progenitor cells was observed when cells were plated at a concentration of 1 cell per well in fetal calf serum-supplemented and serum-depleted medium. Finally, the DNA strand breakage elongation assay showed that Tpo inhibits apoptosis of Lin-Sca-1+ bone marrow cells. Thus, Tpo has a potent ability to promote the viability and suppress apoptosis of primitive multipotent progenitor cells.

Thrombopoietin, a direct stimulator of viability and multilineage growth of primitive bone marrow progenitor cells.

Thrombopoietin (TPO), the ligand for c-mpl, has recently been demonstrated to be the primary regulator of megakaryocytopoiesis and platelet production. In addition, several studies have demonstrated that c-mpl is expressed on hematopoietic cell populations highly enriched in primitive progenitor cells. Here we summarize and discuss recent studies from our laboratory, as well as others, demonstrating that TPO has effects on primitive hematopoietic progenitor cells. When acting alone, TPO stimulates little or no growth, but promotes viability and suppresses apoptosis of murine multipotent (Lin- Sca-1+) bone marrow progenitor cells in vitro. In addition, TPO directly and potently synergizes with other early acting cytokines (kit ligand, flt3 ligand and interleukin 3) to promote multilineage growth of the same progenitor cell population. Although it remains to be established whether TPO also acts on the long-term reconstituting pluripotent stem cells, these studies combined with progenitor cell studies in c-mpl-deficient mice, suggest that TPO, in addition to its key role in platelet production, might also have an important impact on early hematopoiesis.