Enhanced control of proliferation in telomerized cells

Clones of telomerized fibroblasts of adult human skin have earlier been obtained. It was shown that despite their fast growth in mass cultures, these cells poorly form colonies. Conditioned medium, antioxidants, and reduced partial oxygen pressure enhanced their colony formation, but not to the level characteristic of the initial cells. The conditioned medium of telomerized cells enhanced colony formation to a much greater extent than that of the initial cells. A study of proteome of the telomerized fibroblasts has revealed changes in the activities of tens of genes. A general trend consists in weakening and increased lability of the cytoskeleton and in activation of the mechanisms controlling protein degradation. However, these changes are not very pronounced. During the formation of immortal telomerized cells, selection takes place, which appears to determine changes in the expression of some genes. It was proposed that a decrease in the capacity of telomerized cells for colony formation is due to increased requirements of these cells to cell-cell contacts. The rate of cell growth reached that characteristic of mass cultures only in the largest colonies. In this respect, the telomerized fibroblasts resembled stem cells: they are capable of self-maintenance, but "escape" to differentiation in the absence of the corresponding microenvironment (niche), which is represented by other fibroblasts. Non-dividing cells in the test of colony formation should be regarded as differentiated cells, since they have no features of degradation, preserve their viability, actively move, grow, phagocytized debris, etc. It was also shown that telomerization did not prevent differentiation of myoblasts and human neural stem cells. Thus, the results obtained suggest the existence of normal mechanisms underlying the regulation of proliferation in the telomerized cells, which opens possibilities of their use in cell therapy, especially in the case of autotransplantation to senior people, when the cell proliferative potential is markedly reduced and accessibility of stem cells is significantly restricted.     

Permissive role of interleukin 3 (IL-3) in proliferation and differentiation of multipotential hemopoietic progenitors in culture

We studied the effects of interleukin-3 (IL-3) on colony formation by hemopoietic progenitors in methylcellulose cultures of spleen cells from 5-fluorouracil (FU)-treated mice. Purified IL-3 supported the growth of various types of multilineage colonies including blast cell colonies. The types of colonies were similar to those supported by pokeweed-mitogen spleen cell conditioned medium (PWM-SCM), except that IL-3 supported eosinophil and neutrophil expression better. Delayed addition of IL-3 to cultures 7 days after cell plating decreased the number of colonies to one-half the number in cultures with IL-3 added on day 0. It did not alter the proliferative and differentiation characteristics of late emerging multipotential blast cell colonies. These observations suggest that IL-3 does not trigger hemopoietic progenitors into active cell proliferation but is necessary for their continued proliferation. This permissive role of IL-3 is consistent with a stochastic model of stem cell proliferation which features random entry into cell cycle. IL-3 also supported the growth of multilineage colonies from single cells isolated from blast cell colonies by micromanipulation. This result shows that IL-3 acts directly on multipotential progenitors. Analysis of colonies derived from paired progenitors revealed disparate lineage expression and was in accordance with the stochastic model of stem cell differentiation.     

Selection using the alpha-1 integrin (CD49a) enhances the multipotentiality of the mesenchymal stem cell population from heterogeneous bone marrow stromal cells

Bone marrow-derived mesenchymal stem cells consist of a developmentally heterogeneous population of cells obtained from colony forming progenitors. As these colonies express the alpha-1 integrin (CD49a), here we single-cell FACS sorted CD49a+ cells from bone marrow in order to create clones and then compared their colony forming efficiency and multilineage differentiation capacity to the unsorted cells. Following selection, 40% of the sorted CD49a+ cells formed colonies, whereas parental cells failed to form colonies following limited dilution plating at 1 cell/well. Following ex vivo expansion, clones shared a similar morphology to the parental cell line, and also demonstrated enhanced proliferation. Further analysis by flow cytometry using a panel of multilineage markers demonstrated that the CD49a+ clones had enhanced expression of CD90 and CD105 compared to unsorted cells. Culturing cells in adipogenic, osteogenic or chondrogenic medium for 7, 10 and 15 days respectively and then analysing them by quantitative PCR demonstrated that CD49a+ clones readily underwent multlineage differentiation into fat, bone and cartilage compared to unsorted cells. These results thus support the use of CD49a selection for the enrichment of mesenchymal stem cells, and describes a strategy for selecting the most multipotential cells from a heterogeneous pool of bone marrow mononuclear stem cells.     

Characterization of the growth responses of hamster fibroblasts and chondrogenic cells to fibroblast growth factor

The effects of fibroblast growth factor (FGF) on hamster dermal fibroblasts and chondrogenic cells, both of mesodermal origin, were compared with special reference to growth stimulation and morphological changes in monolayer cultures, and colony formation in semisolid medium. FGF (10 to 200 ng/ml) caused appreciable cell proliferation of dermal fibroblasts but not of chondrogenic cells, while FGF (50-200 ng/ml) caused very marked dose-dependent morphological changes in monolayer cultures and colony formation in semisolid medium of both fibroblasts and chondrogenic cells. It is suggested that FGF is the same type of growth factor as the transforming growth factor(s) because, like the latter, it induces drastic morphological changes of normal mesodermal cells in monolayer cultures and their colony formation in semisolid medium.     

无血清无饲养层条件下培养小鼠胚胎干细胞

目的研究在无血清无饲养层条件下小鼠胚胎干细胞的培养方法,为最终建立无血清无饲养层培养系统打下基础。方法比较小鼠胚胎干细胞ES-S8株在无血清培养体系和有血清培养体系中的生长情况,分析ES-S8细胞克隆形成效率,测定其生长速度;然后在撤去血清和饲养层的条件下培养ES-S8细胞,进行AKP染色和表面标记物SSEA-1免疫荧光检测。结果ES-S8细胞在无血清培养条件下细胞生长速度减缓,克隆形成率降低,但AKP染色、SSEA-1免疫荧光均显阳性;在无血清无饲养层条件下ES-S8细胞培养仍能形成克隆,且AKP染色、SSEA-1免疫荧光均显阳性。结论研究表明ES-S8细胞能够在无血清无饲养层的培养条件下生长,保持其良好的未分化特性。     

Colony formation in agar by adult bone marrow multipotential hemopoietic cells

Erythroid colony formation in agar cultures of CBA bone marrow cells was stimulated by the addition of pokeweed mitogen-stimulated spleen conditioned medium (SCM). Optimal colony numbers were obtained when cultures contained 20% fetal calf serum and concentrated spleen conditioned medium. By 7 days of incubation, large burst or unicentric erythroid colonies occurred at a maximum frequency of 40–50 per 10⁵ bone marrow cells. In CBA mice the cells forming erythroid colonies were also present in the spleen, peripheral blood, and within individual spleen colonies. A marked strain variation was noted with CBA mice having the highest levels of erythroid colony-forming cells. In CBA mice erythroid colony-forming cells were mainly non-cycling (12.5% reduction in colony numbers after incubation with hydroxyurea or 3H-thymidine). Erythroid colony-forming cells sedimented with a peak of 4.5 mm/hr, compared with CFU-S, which sedimented at 4.25 mm/hr. The addition of erythropoietin (up to 4 units) to cultures containing SCM did not alter the number or degree of hemoglobinisation of erythroid colonies. Analysis of the total number of erythroid colony-forming cells and CFU-S in 90 individual spleen colonies gave a correlation coefficient of r = 0.93 for these two cell types. In addition to benzidine-positive erythroid cells, up to 40% of the colonies contained, in addition, varying proportions of neutrophils, macrophages, eosinophils, and megakaryocytes. Taken together with the close correlation between the numbers of CFU-S in different adult hemopoietic tissues, including individual spleen colonies, the data indicate that the erythroid colony-forming cells expressing multiple hemopoietic differentiation are members of the hemopoietic multipotential stem cell compartment.     

The enhancement of in vitro survival and chondrogenesis of limb bud cells by cartilage conditioned medium

Dissociated stage 21–28 chick embryo limb bud cells showed an increasing ability to produce cartilage colonies in vitro with in vivo maturation. In addition dissociated stage 21–28 chick embryo limb bud cells exposed to cartilage conditioned medium continuously or only for 48 hr prior to subculture showed an enhanced (as much as 15-fold) ability to form differentiated cartilage colonies. By this criterion, cells were more responsive to conditioned medium prior to stage 25. Conditioned medium from fibroblast cultures caused an inhibition of cartilage colony formation, suggesting that the effect is cell-type specific. Besides increasing cartilage colony formation by enhanced cell survival, the incorporation of S³⁵O₄ into isolated glycosaminoglycans is also stimulated when limb bud cells are exposed to cartilage conditioned medium. The results support a model for cell differentiation which involves the enhancement of a particular differentiated capacity by a diffusible cell-type-specific macromolecule.     

A factor produced by human cells in vitro that changes HeLa cell colonial morphology

Summary Colonies of HeLa cells cultured in media supplemented with human or bovine serum or both can be morphologically described as three types: diffuse, intermediate, and compact, with their modal distribution depending on the serum or sera added to the growth medium. We have observed that for a particular medium or serum system, the percentage of compact colonies remains fairly constant under normal culture conditions, 0.2%, whereas the diffuse and intermediate colonies vary over a much wider range. The presence of certain substances as trypsin, heparin and Darvan in the medium favor the increase of compact colonies at the expense of other types. Furthermore, we have discovered that colonial morphology is influenced by cocultivation of the HeLa cells with human fibroblastlike cells, the compact colonies increasing as the density of the fibroblast element introduced into the mixed cultures is increased. Subsequent investigation revealed that conditioned medium from confluent fibroblast and HeLa cell cultures contained a factor(s), that significantly increased the percentage of compact colonies. The factor is nondialyzable, heat-stable and can be neutralized by serum. Recorded in this presentation are preliminary observations on the kinetics of colony formation and the interaction among the three HeLa cell colony types, the diffuse, the intermediate, and the compact. The factor's effect on HeLa cell colonial morphology is time dependent and rapidly reversed if the factor(s) is removed and fresh medium added.     

In vitro inhibitory effect of interferon on colony formation of myeloma stem cells

Summary The inhibitory effect of interferon on colony formation of myeloma stem cells in two layer plasma clot-soft agar cultures was studied. Human lymphoblast interferon inhibited in therapeutically attainable concentrations myeloma stem cell proliferation in 50% and human fibroblast interferon in 23% of the 14 myeloma patients in whom in vitro colony formation could be achieved. In interferon-sensitive patients the numbers of myeloma stem cell clusters and colonies were decreased to 34.4%–54.9% of control cultures. In addition, maturation of myeloma stem cells in differentiated plasma cells was reduced by interferon in most of these cases.     

Adipose conversion of mouse bone marrow fibroblasts in vitro: their alkaline phosphatase activity

By an in vitro colony assay and cytochemical staining, we investigated the capacity of mouse bone marrow fibroblasts to differentiate into adipocytes and to express alkaline phosphatase (ALP) activity. Glucocorticoids enhanced colony formation of the fibroblasts, and stimulated their adipose conversion (55-65% of the colonies became adipocyte-positive), but they did not affect ALP activity. The fibroblasts became heterogeneous in size and morphology after growing in vitro then differentiated into adipocytes. All the cell types had ALP activity, and more than 95% of the colonies contained ALP-positive cells. ALP staining was strongest in cells in the early stage of adipose conversion, gradually decreasing with maturation. Our results indicate that the majority of the mouse bone marrow fibroblasts that formed colonies under our culture conditions are preadipocytes. We conclude that these fibroblasts originate from adventitial reticular cells present in bone marrow stroma because reticular cells have been reported to possess high ALP activity and have been suggested to differentiate into adipocytes.     

Differentiation of Mouse Bone Marrow Precursor Cells into Neutrophil Granulocytes by an Activity Separation from WEHI-3 Cell-Conditioned Medium

Colonies comprised exclusively of neutrophil granulocytes have been obtained by growing mouse bone marrow cells in nutrient semisolid agar cultures. A stimulator of predominantly granulocyte colony formation was present in the breakthrough fraction of preparations of colony-stimulating activity separated on DEAE-Sephadex A. The source of colony-stimulating activity was concentrated conditioned medium of a murine myelomonocytic cell line (WEHI-3), which unfractionated stimulated the growth of colonies of granulocytes, macrophages, megakaryocytes, as well as mixed colony types. After stepwise column chromatography of the conditioned medium, the breakthrough fraction was shown to stimulate predominantly granulocyte colony formation, and the fraction eluted with 1 M NaCl was found to induce primarily macrophage colony growth. Colony morphology was independent of the concentration of eluate used. The morphology of colonies varied with increasing concentrations of the breakthrough fraction. At low concentrations, granulocyte colony formation was almost exclusively observed. With increasing concentrations of this fraction, an increasing proportion of the colonies were found to contain macrophages. The effect of concentration of this activity was in marked contrast to previous findings where the incidence of granulocyte colony formation was inversely related to the concentration of colony-stimulating activity. This differential responsiveness of cell to stimulus has previously been interpreted as low concentrations of a growth and differentiation factor being required for macrophage production and high concentrations of the same factor required for granulocyte formation. Separation of these activities by DEAE Sephadex chromatography, and alteration of the dose-response curve, such that granulocyte colony formation varies directly with the amount of stimulator, indicates that the differentiation of these two cell blood lineages may be controlled by separate entities.     

Noggin maintains pluripotency of human embryonic stem cells grown on Matrigel

Objective:  Spontaneous differentiation of human embryonic stem cell (hESC) cultures is a major concern in stem cell research. Physical removal of differentiated areas in a stem cell colony is the current approach used to keep the cultures in a pluripotent state for a prolonged period of time. All hESCs available for research require unidentified soluble factors secreted from feeder layers to maintain the undifferentiated state and pluripotency. Under experimental conditions, stem cells are grown on various matrices, the most commonly used being Matrigel.
Materials and Methods:  We propose an alternative method to prevent spontaneous differentiation of hESCs grown on Matrigel that uses low amounts of recombinant noggin. We make use of the porosity of Matrigel to serve as a matrix that traps noggin and gradually releases it into the culture to antagonize bone morphogenetic proteins (BMP). BMPs are known to initiate differentiation of hESCs and are either present in the conditioned medium or are secreted by hESCs themselves.
Results:  hESCs grown on Matrigel supplemented with noggin in conditioned medium from feeder layers (irradiated mouse embryonic fibroblasts) retained both normal karyotype and markers of hESC pluripotency for 14 days. In addition, these cultures were found to have increased cell proliferation of stem cells as compared to hESCs grown on Matrigel alone.
Conclusion:  Noggin can be utilized for short term prevention of spontaneous differentiation of stem cells grown on Matrigel.     

Opposing effects of 12-o-tetradecanoylphorbol 13-acetate on human myeloid and lymphoid cell proliferation

The effect of 12-O-tetradecanoylphorbol 13-acetate (TPA) on human hematopoietic cells has been investigated. It was found that 1–10 ng/ml of TPA totally abrogated erythroid and granulocytic colony growth and, simultaneously in the presence of PHA, stimulated T-lymphocyte colony formation. TPA concentrations insufficient to inhibit myeloid colony growth also failed to stimulate lymphocyte colony formation. Optimal culture conditions for the growth of these colonies required the presence of TPA, PHA, and leukocyte-conditioned medium in the cultures. Cells within the colonies were 80–90% E-rosette positive and by monoclonal antibody characterization contained 45–66% OKT3-positive cells. Colony-forming cells were found in both E-rosette-positive and-negative fractions. Although by cell surface marker characterization the cells within the colonies had properties of T-cells, the exact relationship of cells forming colonies under these conditions to those detected in other T-cell colony assays remain to be determined.     

Self-renewal and commitment to differentiation of human leukemic promyelocytic cells (HL-60)

More than 80% of cells from a human promyelocytic leukemic cell line (HL-60) possess the capacity for self-renewal as evidenced by their ability to form large primary colonies in semisolid medium and the presence within these colonies of cells capable of subsequent colony formation. Colony development is independent of the normal regulator-the myeloid colony stimulating factor. The observed autostimulation suggests the production of specific growth promoters by the cells. Differentiation either to mature granulocytes or macrophages, induced by various agents, was associated with reduced cloning potential. Nevertheless, colonies containing differentiated cells could be developed either by cloning cells in the presence of suboptimal concentrations of inducer or by adding inducers over colonies developed in its absence. Upon differentiation, there was a morphological change from compact to diffused colony morphology due to cell mobility in the semisolid medium. Even at suboptimal concentrations of inducer more than 95% of the colonies became diffused, indicating clonaI homogeneity of the population with respect to differentiation capacity. The loss of self-renewal was found to be one of the early properties which changed following the initiation of differentiation. The loss preceded not only the overt expression of maturation-specific functions but also cellular commitment to terminal differentiation; shorter contact with the inducer was required to cause loss of self-renewal than to induce an irreversible transition to differentiation. This resulted in cells that lost their self-renewal potential without being able to complete their program of differentiation.     

FGF4-dependent stem cells derived from rat blastocysts differentiate along the trophoblast lineage

Differentiated trophoblast cell lineages arise from trophoblast stem (TS) cells. To date such a stem cell population has only been established in the mouse. The objective of this investigation was to establish TS cell populations from rat blastocysts. Blastocysts were cultured individually on a feeder layer of rat embryonic fibroblasts (REFs) in fibroblast growth factor-4 (FGF4) and heparin supplemented culture medium. Once cell colonies were established REF feeder layers could be replaced with REF conditioned medium. The blastocyst-derived cell lines, in either proliferative or differentiated states, did not express genes indicative of ICM-derived tissues. In the proliferative state the cells expressed established stem cell-associated markers of TS cells. Cells ceased proliferation and differentiated when FGF4, heparin, and REF conditioned medium were removed. Differentiation was characterized by a decline of stem cell-associated marker gene expression, the appearance of large polyploid cells (trophoblast giant cells), and the expression of trophoblast differentiation-associated genes. Collectively, the data indicate that the rat blastocyst-derived cell lines not only possess many features characteristic of mouse TS cells but also possess some distinct properties. These rat TS cell lines represent valuable new in vitro models for analyses of mechanisms controlling TS cell renewal and differentiation.     

CFU-F circulating in cord blood

CFU-F (colony forming units-fibroblast) were studied from cord blood and, as controls, from normal bone marrow of older children and adults. Numbers of CFU-F in cord blood buffy coat cells are lower by a factor of 10 in comparison to bone marrow CFU-F. Cytomorphology and staining with monoclonal antibody identify the progeny cells of CFU-F as fibroblasts. Cord blood CFU-F derived fibroblasts have properties supporting hematopoiesis: They produce CSF (colony stimulating factor) to which fresh cord blood CFU-GM (colony forming units-granulocytic, monocytic) react by colony formation in a dose-response manner. In addition, fibroblast colonies discharge clonogenic round cells into the medium forming CFU-GM and CFU-F colonies in secondary methyl cellulose cultures. We conclude that fetal blood contains clonogenic stromal cells (CFU-F) that give rise to fibroblasts with properties of hematopoietic support.     

Isolation and characterization of embryonic stem cell-like cells from in vitro-produced buffalo (Bubalus bubalis) embryos

This study was carried out to isolate and characterize buffalo embryonic stem (ES) cell-like cells from in vitro-produced embryos. Inner cell mass (ICM) cells were isolated either mechanically or by enzymatic digestion from 120 blastocysts whereas 28 morulae were used for the isolation of blastomeres mechanically. The ICM cells/ blastomeres were cultured on mitomycin-C-treated feeder layer. Primary cell colony formation was higher (P < 0.05) for hatched blastocysts (73.1%, 30/41) than that for early/expanded blastocysts (25.3%, 20/79). However, no primary cell colonies were formed when blastomeres obtained from morulae were cultured. Primary colonies were formed in 14.1% (12/85) of intact blastocyst culture, which was significantly lower (P < 0.05) than that of 41.6% for ICM culture. These colonies were separated by enzymatic or mechanical disaggregation. Using mechanical disaggregation method, the cells remained undifferentiated and two buffalo ES cell-like cell lines (bES1, bES2) continued to grow in culture up to eight passages. However, disassociation through enzymatic method resulted in differentiation. Undifferentiated cells exhibited stem cell morphological features, normal chromosomal morphology, and expressed specific markers such as alkaline phosphatase (AP) and Oct-4. Cells formed embryoid bodies (EBs) in suspension culture; extended culture of EBs resulted in formation of cystic EBs. Following prolonged in vitro culture, these cells differentiated into several types of cells including neuron-like and epithelium-like cells. Furthermore, the vitrified-thawed ES cell-like cells also exhibited typical stem cell characteristics. In conclusion, buffalo ES cell-like cells could be isolated from in vitro-produced blastocysts and maintained in vitro for prolonged periods of time.