Presence Of Feeder Cells Research Articles

Phenotype of NK cells after in vitro cultivation in the presence of feeder cells

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Activation of NK cells in bioreactor systems after in vitro cultivation in the presence of feeder cells

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Butyrate and propionate regulated proliferation and activation of human group–2 innate lymphoid cells (ILC2s)

Commensal intestinal microbiota are thought to play fundamental roles in the induction, training and regulation of the human immune system, at least in part through innumerable microbial products such as short-chain fatty acids (SCFAs). We hypothesized that SCFAs such as butyrate, propionate and acetate directly affect group-2 innate lymphoid cell (ILC2) functions and regulate allergic inflammation. To test this hypothesis, we determined the effects of SCFAs on human ILC2s in vitro. Human ILC2s were sorted from peripheral blood lymphocytes (PBMC) of healthy donors and expanded in the presence of feeder cells and recombinant IL-2 for 3-4 weeks. After confirming their purity, the ILC2s were cultured with IL-2 and IL-33 in the presence and absence of such SCFAs as butyrate, propionate and acetate. Then the cells’ viability, proliferation and cytokine/chemokine production were determined. We found that butyrate and propionate suppressed the ILC2s’ production of such type 2 cytokines/chemokine as IL-5, IL-9, IL-13 and IL-8 . Butyrate and propionate also induced apoptosis and suppressed proliferation of the cells through downregulation of IL-2Rα. Conversely, acetate showed none of those effects on the cells. Butyrate and propionate, but not acetate, produced by the intestinal microbiota may play important roles in regulating the proliferation activation and cytokine/chemokine production profiles of ILC2s, thereby leading to regulation of allergic inflammatory diseases.

Conditionally reprogrammed normal and primary tumor prostate epithelial cells: a novel patient-derived cell model for studies of human prostate cancer.

Our previous study demonstrated that conditional reprogramming (CR) allows the establishment of patient-derived normal and tumor epithelial cell cultures from a variety of tissue types including breast, lung, colon and prostate. Using CR, we have established matched normal and tumor cultures, GUMC-29 and GUMC-30 respectively, from a patient's prostatectomy specimen. These CR cells proliferate indefinitely in vitro and retain stable karyotypes. Most importantly, only tumor-derived CR cells (GUMC-30) produced tumors in xenografted SCID mice, demonstrating maintenance of the critical tumor phenotype. Characterization of cells with DNA fingerprinting demonstrated identical patterns in normal and tumor CR cells as well as in xenografted tumors. By flow cytometry, both normal and tumor CR cells expressed basal, luminal, and stem cell markers, with the majority of the normal and tumor CR cells expressing prostate basal cell markers, CD44 and Trop2, as well as luminal marker, CD13, suggesting a transit-amplifying phenotype. Consistent with this phenotype, real time RT-PCR analyses demonstrated that CR cells predominantly expressed high levels of basal cell markers (KRT5, KRT14 and p63), and low levels of luminal markers. When the CR tumor cells were injected into SCID mice, the expression of luminal markers (AR, NKX3.1) increased significantly, while basal cell markers dramatically decreased. These data suggest that CR cells maintain high levels of proliferation and low levels of differentiation in the presence of feeder cells and ROCK inhibitor, but undergo differentiation once injected into SCID mice. Genomic analyses, including SNP and INDEL, identified genes mutated in tumor cells, including components of apoptosis, cell attachment, and hypoxia pathways. The use of matched patient-derived cells provides a unique in vitro model for studies of early prostate cancer.

Conserved Role of bFGF and a Divergent Role of LIF for Pluripotency Maintenance and Survival in Canine Pluripotent Stem Cells.

Dogs have been widely used as a preclinical model for human disease. With the successful generation of canine induced pluripotent stem cells (ciPSCs), the biomedical community has a unique opportunity to study therapeutic interventions using autologous stem cells that can benefit dogs and humans. Unlike mice and human pluripotent cells, which are leukemia inhibitory factor (LIF)- and basic fibroblast growth factor (bFGF)-dependent, respectively, dog iPSCs require both growth factors simultaneously. In an effort to elucidate the role of each factor in the control of ciPSC self-renewal, we performed a series of experiments aiming at understanding the signaling pathways activated by them. We found that bFGF regulates pluripotency by indirectly activating the SMAD2/3 pathway in the presence of feeder cells, exclusively targeting NANOG expression, and inhibiting spontaneous differentiation toward ectoderm and mesoderm. LIF activates the JAK-STAT3 pathway but does not function in the typical manner described in mouse naïve embryonic stem cells. These results show that a unique mechanism for maintenance of pluripotency is present in ciPSC. These findings should be taken into account when establishing stem cell differentiation protocols and may provide more insight into pluripotency regulation in species other than mice and humans.

Treatment of Silk Fibroin with Poly(ethylene glycol) for the Enhancement of Corneal Epithelial Cell Growth.

A silk protein, fibroin, was isolated from the cocoons of the domesticated silkworm (Bombyx mori) and cast into membranes to serve as freestanding templates for tissue-engineered corneal cell constructs to be used in ocular surface reconstruction. In this study, we sought to enhance the attachment and proliferation of corneal epithelial cells by increasing the permeability of the fibroin membranes and the topographic roughness of their surface. By mixing the fibroin solution with poly(ethylene glycol) (PEG) of molecular weight 300 Da, membranes were produced with increased permeability and with topographic patterns generated on their surface. In order to enhance their mechanical stability, some PEG-treated membranes were also crosslinked with genipin. The resulting membranes were thoroughly characterized and compared to the non-treated membranes. The PEG-treated membranes were similar in tensile strength to the non-treated ones, but their elastic modulus was higher and elongation lower, indicating enhanced rigidity. The crosslinking with genipin did not induce a significant improvement in mechanical properties. In cultures of a human-derived corneal epithelial cell line (HCE-T), the PEG treatment of the substratum did not improve the attachment of cells and it enhanced only slightly the cell proliferation in the longer term. Likewise, primary cultures of human limbal epithelial cells grew equally well on both non-treated and PEG-treated membranes, and the stratification of cultures was consistently improved in the presence of an underlying culture of irradiated 3T3 feeder cells, irrespectively of PEG-treatment. Nevertheless, the cultures grown on the PEG-treated membranes in the presence of feeder cells did display a higher nuclear-to-cytoplasmic ratio suggesting a more proliferative phenotype. We concluded that while the treatment with PEG had a significant effect on some structural properties of the B. mori silk fibroin (BMSF) membranes, there were minimal gains in the performance of these materials as a substratum for corneal epithelial cell growth. The reduced mechanical stability of freestanding PEG-treated membranes makes them a less viable choice than the non-treated membranes.

A novel feeder-free culture system for expansion of mouse spermatogonial stem cells.

Spermatogonial stem cells (SSCs, also called germline stem cells) are self-renewing unipotent stem cells that produce differentiating germ cells in the testis. SSCs can be isolated from the testis and cultured in vitro for long-term periods in the presence of feeder cells (often mouse embryonic fibroblasts). However, the maintenance of SSC feeder culture systems is tedious because preparation of feeder cells is needed at each subculture. In this study, we developed a Matrigel-based feeder-free culture system for long-term propagation of SSCs. Although several in vitro SSC culture systems without feeder cells have been previously described, our Matrigel-based feeder-free culture system is time- and cost- effective, and preserves self-renewability of SSCs. In addition, the growth rate of SSCs cultured using our newly developed system is equivalent to that in feeder cultures. We confirmed that the feeder-free cultured SSCs expressed germ cell markers both at the mRNA and protein levels. Furthermore, the functionality of feeder-free cultured SSCs was confirmed by their transplantation into germ cell-depleted mice. These results suggest that our newly developed feeder-free culture system provides a simple approach to maintaining SSCs in vitro and studying the basic biology of SSCs, including determination of their fate.

Combining ABL1 Kinase Inhibitor, Imatinib and the Jak Kinase Inhibitor TG101348: A Potential Treatment for Residual BCR-ABL Positive Leukemia Cells

Abstract 3737ABL kinase inhibitor, imatinib is highly effective therapy against chronic myeloid leukemia (CML) patients and eliminates disease progression and transformation. However, imatinib is not curative for most CML patients. Residual CML cells are present in bone marrow microenvironment. Bone marrow microenvironment is a source of soluble factors and regulates the proliferation of leukemia cells. These leukemia cells are contained within a niche in the bone marrow and are often impervious to current treatments, thus maintaining their proliferative activity when the treatment is ceased, suggests that the new therapeutic strategies designed to override stroma-associated drug resistance are required to treat against Philadelphia (Ph)-positive leukemia patients. The hematopoietic cytokine receptor signaling is mediated by tyrosine kinases termed Janus kinases (Jaks) and downstream transcription factors, signal transducers and activators of transcription (STATs). Jak-STAT signaling is also activated in CML cells. One of the Jak kinase inhibitor, TG101348 (SAR302503) is an orally available inhibitor of Jak2 and developed for the treatment of patients with myeloproliferative diseases. Therefore, combination therapy using a BCR-ABL tyrosine kinase inhibitors and a Jak inhibitor, TG101348 may help prevent stroma-associated drug resistance and these approaches may be expected to improve the outcomes of CML patients. In this study, we investigated the ABL tyrosine kinase inhibitor, imatinib and TG101348 efficacy by using the BCR-ABL positive cell lines, K562 and primary CML samples when leukemic cells were protected by the feeder cell lines (HS-5 and S9). 72 hours treatment of imatinib exhibits cell growth inhibition and induced apoptosis against K562 cells in a dose dependent manner. However, the treatment of imatinib exhibits cell growth inhibition partially against K562 cells in the presence of HS-5 conditioned media. We found that the treatment of TG101348 did not exhibit cell growth inhibition against K562 cells directly, but the combination treatment with imatinib and TG101348 abrogated the protective effects of HS-5 conditioned media in K562 cells. We next investigated the intracellular signaling of imatinib and TG101348. Phosphorylation of BCR-ABL, Crk-L was not reduced after TG101348 treatment. However, phosphorylation of BCR-ABL, Crk-L was significantly reduced and increased apoptosis after combination treatment with imatinib and TG101348. We next investigated the efficacy between imatinib and TG101348 by using CD34 positive primary CML samples. The treatment of imatinib exhibits cell growth inhibition partially against CD34 positive CML samples in the presence of feeder cells. Combined treatment of CD34 positive primary samples with imatinib and TG101348 caused significantly more cytotoxicity and induced apoptosis. We also found that mitogen-activated protein kinase (MAPK) was also inhibited by imatinib and TG101348 treatment. We next investigated the intracellular signaling of imatinib and TG101348 by using the CD34 positive primary samples. Phosphorylation of BCR-ABL, Crk-L was significantly reduced and increased apoptosis after treatment with imatinib and TG101348. Moreover, combination of imatinib and TG101348 inhibited the colony growth of Ph-positive primary samples. We also investigated the TG101348 activity against feeder cell. Phosphorylation of STAT5 was reduced by TG101348 in a dose dependent manner. The cytokine production was analyzed by using cytokine array systems. The cytokine production such as granulocyte macrophage colony-stimulating factor (GM-CSF) from HS-5 was also reduced by TG101348 treatment. Data from this study suggested that administration of the imatinib and Jak inhibitor, TG101348 may be a powerful strategy against stroma-associated drug resistance of Ph-positive cells and enhance cytotoxic effects of imatinib in those residual CML cells. Disclosures:No relevant conflicts of interest to declare.

Dynamic Status of REST in the Mouse ESC Pluripotency Network

BackgroundREST is abundantly expressed in mouse embryonic stem cells (ESCs). Many genome-wide analyses have found REST to be an integral part of the ESC pluripotency network. However, experimental systems have produced contradictory findings: (1) REST is required for the maintenance of ESC pluripotency and loss of REST causes increased expression of differentiation markers, (2) REST is not required for the maintenance of ESC pluripotency and loss of REST does not change expression of differentiation markers, and (3) REST is not required for the maintenance of ESC pluripotency but loss of REST causes decreased expression of differentiation markers. These reports highlight gaps in our knowledge of the ESC network.MethodsEmploying biochemical and genome-wide analyses of various culture conditions and ESC lines, we have attempted to resolve some of the discrepancies in the literature.ResultsWe show that Rest+/− and Rest−/− AB-1 mutant ESCs, which did not exhibit a role of REST in ESC pluripotency when cultured in the presence of feeder cells, did show impaired self-renewal when compared with the parental cells under feeder-free culture conditions, but only in early passage cells. In late passage cells, both Rest+/− and Rest−/− AB-1 ESCs restored pluripotency, suggesting a passage and culture condition-dependent response. Genome-wide analysis followed by biochemical validation supported this response and further indicated that the restoration of pluripotency was associated by increased expression of the ESC pluripotency factors. E14Tg2a.4 ESCs with REST-knockdown, which earlier showed a REST-dependent pluripotency when cultured under feeder-free conditions, as well as Rest−/− AB-1 ESCs, showed no REST-dependent pluripotency when cultured in the presence of either feeder cells or laminin, indicating that extracellular matrix components can rescue REST's role in ESC pluripotency.ConclusionsREST regulates ESC pluripotency in culture condition- and ESC line-dependent fashion and ESC pluripotency needs to be evaluated in a context dependent manner.

Effects of the Hedgehog Inhibitor GDC-0449, Alone or in Combination with Dasatinib, on BCR-ABL-Positive Leukemia Cells

Hedgehog (Hh)-glioma-associated oncogene homolog (Gli) signaling is implicated in a large number of human cancers such as leukemia. In this study, we investigated the effects of the potent Hh antagonist GDC-0449 on the BCR-ABL-positive cell line OM9;22 and primary samples when leukemia cells were protected by a feeder cell line (S9 cells). The numbers of OM9;22 cells significantly increased with S9 cells. Treatment of OM9;22 cells with GDC-0449 caused cell growth inhibition and induced apoptosis. Moreover, GDC-0449 inhibited the colony growth of Philadelphia chromosome (Ph)-positive primary samples. We next investigated the effects of a combination of GDC-0449 and dasatinib on these cell lines. The growth inhibition typically promoted by dasatinib was significantly reduced in the presence of S9 cells. Treatment of Ph-positive leukemia cells with GDC-0449 and dasatinib in the presence of S9 caused significantly more cytotoxicity than that caused by each drug alone. Inhibition of Gli1 or Gli2 by siRNA transfection reduced the growth of the Ph-positive cell line K562 and increased cytotoxicity of dasatinib. Moreover, colony formations of Gli1 or Gli2 knockdown cells were also reduced. Data from this study suggest that administration of the Hh inhibitor GDC-0449 inhibits BCR-ABL-positive cell growth and enhances the cytotoxic effects of dasatinib in the presence of feeder cells.

Generation of Pig iPS Cells: A Model for Cell Therapy

Reprogramming of pig somatic cells to induced pluripotent stem cells provides a tremendous advance in the field of regenerative medicine since the pig represents an ideal large animal model for the preclinical testing of emerging cell therapies. However, the current generation of pig-induced pluripotent stem cells (piPSCs) require the use of time-consuming and laborious retroviral or lentiviral transduction approaches, in order to ectopically express the pluripotency-associated transcription factors Oct4, Sox2, Klf4 and c-Myc, in the presence of feeder cells. Here, we describe a simple method to produce piPSC with a single transfection of a CAG-driven polycistronic plasmid expressing Oct4, Sox2, Klf4, c-Myc and a green fluorescent protein (GFP) reporter gene, in gelatine-coated plates, with or without feeder cells. In our system, the derivation of piPSCs from adult pig ear fibroblasts on a gelatine coating showed a higher efficiency and rate of reprogramming when compared with three consecutive retroviral transductions of a similar polycistronic construct. Our piPSCs expressed the classical embryonic stem cell markers, exhibit a stable karyotype and formed teratomas. Moreover, we also developed a simple method to generate in vitro spontaneous beating cardiomiocyte-like cells from piPSCs. Overall, our preliminary results set the bases for the massive production of xeno-free and integration-free piPSCs and provide a powerful tool for the preclinical application of iPSC technology in a large animal setting.

Autonomous Regulation of Sex-Specific Developmental Programming in Mouse Fetal Germ Cells1

In mice, unique events regulating epigenetic programming (e.g., genomic imprinting) and replication state (mitosis versus meiosis) occur during fetal germ cell development. To determine whether these processes are autonomously programmed in fetal germ cells or are dependent upon ongoing instructive interactions with surrounding gonadal somatic cells, we isolated male and female germ cells at 13.5 days postcoitum (dpc) and maintained them in culture for 6 days, either alone or in the presence of feeder cells or gonadal somatic cells. We examined allele-specific DNA methylation in the imprinted H19 and Snrpn genes, and we also determined whether these cells remained mitotic or entered meiosis. Our results show that isolated male germ cells are able to establish a characteristic "paternal" methylation pattern at imprinted genes in the absence of any support from somatic cells. On the other hand, cultured female germ cells maintain a hypomethylated status at these loci, characteristic of the normal "maternal" methylation pattern in endogenous female germ cells before birth. Further, the surviving female germ cells entered first meiotic prophase and reached the pachytene stage, whereas male germ cells entered mitotic arrest. These results indicate that mechanisms controlling both epigenetic programming and replication state are autonomously regulated in fetal germ cells that have been exposed to the genital ridge prior to 13.5 dpc.

Embryonic Stem Cells

Stem cells are unique cell populations with the ability to choose between self-renewal and differentiation. Embryonic stem (ES) cells have the ability to form any fully differentiated cell of the body. To date, only three species of mammals have yielded long-term cultures of self-renewing ES cells- mice, monkeys, and humans. These cells have some special requirements to maintain their undifferentiated state in culture, e.g., presence of feeder cells, serum, or cytokines. Many scientific studies have tried to manipulate the growth and differentiation conditions with varied success. Studies of development in model systems, such as mice help our efforts to manipulate human stem cells in vitro. Data are now emerging that ES cells can be directed toward lineage-specific differentiation programs. On the basis of this property, it is likely that human ES cells will provide a useful differentiation culture system to study the mechanisms of human development. Recent advances in culturing ES cells and success in exploiting their pluripotency brings great hope for using human ES cell-based reparative therapy in future.

Immunocyto- and histochemical profiling of nucleostemin expression: Marker of epidermal stem cells?

Because the nucleolar protein nucleostemin is present in bone marrow and neuronal stem cells and malignancies originating thereof we monitored its expression in frozen sections from normal human epidermis, basal cell carcinomas, cultured keratinocytes and cells of the squamous carcinoma line FaDu. In addition, probing the value of this protein as a marker of epidermal stem cells was an aim of this study. To further characterize cell features we added analysis of expression of keratins 10 or 19 as markers of terminal differentiation and Ki67 as marker of proliferating cells as well as three adhesion/growth-regulatory galectins. Immunohistochemical monitoring revealed expression of nucleostemin in cells of both Ki67-positive and -negative nuclei regardless of the K10-expression status. Cultured keratinocytes were positive, when they were prepared from hair follicles and cultured in the presence of feeder cells. A small population of these nucleostemin-positive cells also expressed galectin-1 but not galectins-3 and -9 in their nucleoli. Part of these cells also expressed keratin 19. FaDu cells were strongly positive, illustrating expression in malignant cells which require no feeder layer. Of note, the number of galectin-1-positive nucleoli was reduced in the course of culture. Nucleostemin positivity cannot be considered as marker for stem cells in skin sections. In cultured cells, nucleostemin is expressed in a distinct population of the epidermal cells from hair follicle kept in the presence of a feeder layer, intimating an association of nucleostemin expression with this type of epithelio-mesenchymal interaction which is not essential during propagation of malignant cells.

Methylation of the p16INK4a promoter region in telomerase immortalized human keratinocytes co-cultured with feeder cells

Human keratinocytes grown in co-culture with fibroblast feeder cells have an extended in vitro lifespan and delayed accumulation of the tumor suppressor protein p16(INK4a) when compared to the same cells grown on tissue culture plastic alone. Previous studies have indicated that human keratinocytes can be immortalized by telomerase activity alone when grown in co-culture with feeder cells, suggesting that loss of the p16(INK4a)/Rb pathway is not required for immortalization. Using two independent human keratinocyte cell strains, we found that exogenous telomerase expression and co-culture with feeder cells results in efficient extension of lifespan without an apparent crisis. However, when these cells were transferred from the co-culture environment to plastic alone they experienced only a brief period of slowed growth before continuing to proliferate indefinitely. Examination of immortal cell lines demonstrated p16(INK4a) promoter methylation had occurred in both the absence and presence of feeder cells. Reintroduction of p16(INK4a) into immortal cell lines resulted in rapid growth arrest. Our results suggest that p16(INK4a)/Rb-induced telomere-independent senescence, although delayed in the presence of feeders, still provides a proliferation barrier to human keratinocytes in this culture system and that extended culture of telomerase-transduced keratinocytes on feeders can lead to the methylation of p16(INK4a).

Telomerase‐specific T‐cells kill pancreatic tumor cells in vitro and in vivo

Adoptive cell transfer is described as an innovative and challenging option for the treatment of malignant melanoma. In the current study, the generation and expansion of telomerase-specific T-cells for adoptive cell transfer and their use in a syngeneic pancreatic carcinoma mouse model was investigated. Telomerase-specific T-cells were generated either in vitro by coculture of human lymphocytes with telomerase-peptide-pulsed dendritic cells or in vivo by injection of peptide plus adjuvant into C57BL/6 mice. Spleens were harvested after immunization and lymphocytes were expanded in the presence of feeder cells. T-cells were tested in vitro against human leukocyte antigen (HLA)-matched, telomerase-positive pancreatic carcinoma cells. Tumor-bearing (subcutaneous) mice pretreated with cyclophosphamide were injected intravenously with the expanded cells. It was possible to generate and expand telomerase-specific T-cells with cytotoxic activity. The protocol did not work as well in the murine setting. However, adoptive cell transfer with murine antigen-specific T-cells delayed disease progression in tumor-bearing mice significantly. Generation of antigen-specific T-cells is feasible; the expansion of these cells could be accomplished without loss of function. Antigen-specific T-cells demonstrated significant cytotoxic activity in a syngeneic, subcutaneous mouse model. However, further optimization of the expansion protocol is warranted.

Culture of Human Embryonic Stem Cells and the Extracellular Matrix Microenvironment

Among the major obstacles impeding successful derivation and continuous culture of human embryonic stem cells (hESC) for therapeutic purposes, are the presence of feeder cells and feeder-conditioned media of animal origin. The risk of contamination with xenopathogens makes hESC cultured in this way unsafe for future use in regenerative medicine. A holy grail for investigators in the field will be to establish and maintain new hESC lines in completely feeder-free and serum-free defined conditions. Recently, propagation of hESC has become possible, using mammalian- or human-derived extracellular matrix (ECM) and conditioned medium from feeder cells. In addition, providing a three-dimensional ECM environment can even support the derivation of new hESC. In this review, we examine recent advances in the use and development of substrates suitable for the derivation and maintenance of hESC, and our current understanding of the effects of a three-dimensional ECM milieu on cellular behavior.

Comparative phenotypic characterization of keratinocytes originating from hair follicles

The principal pool of epidermal stem cells is located in the bulge region of the hair follicle root sheath. In this research project, we have used a refined procedure to isolate porcine hair follicles including their root sheath and for comparison purposes also human cell material. These cells migrating from the hair follicles were then cytochemically characterized. A panel of antibodies and two labeled plant lectins were tested on cell material obtained under a range of assorted experimental conditions. Due to their role in growth regulation we also studied two endogenous lectins, specifically monitoring their expression and the presence of accessible ligands. These in vitro results were compared with findings on porcine and human hair follicles and human basal cell carcinomas in situ. The keratinocytes originating from hair follicles in the presence of feeder cells are rather undifferentiated and express galectin-1/galectin-1-binding sites but not galectin-3 in their nuclei associated with DeltaNp63alpha positivity. Nuclear reactivity for galectin-1 was rarely observed in the bulge of the outer root sheath of the human hair follicle and of basal cell carcinomas and absent in porcine tissue samples. Exclusion of feeder cells from our cultivation system of porcine hair follicles led to the formation of spheroid bodies from these keratinocytes. Ki67 as a marker of proliferation was not present in the nuclei of cells forming these spheroids. One part of these bodies is positive for markers of post-mitotic differentiated cells, while the other spheroids are composed of poorly differentiated cells, which are able to adhere to feeder cells and form growing colonies. In summary, the detection of galectin-1 and also nuclear binding sites for this endogenous effector points to intracellular functionality of this lectin. It can be considered a potential marker of a distinct cell population, probably at the beginning of a differentiation cascade of keratinocytes.

174 ISOLATION, CULTURE AND POTENTIAL USE OF THE PORCINE NEURAL AND EPIDERMAL STEM CELLS

The mammalian brain and epidermis contain stem cells, so-called neural stem cells (NSC) and epidermal stem cells (EpSC). To achieve the full therapeutic potential of stem cells, appropriate animal models have to be used to establish the sequence of pathological changes and to test potential therapies to block these changes. In the following studies miniature pigs were used as a biomedical model. We isolated multipotent cells from brains of porcine fetuses for future use in allotransplantation experiments in the inbred miniature pig strain. Brain tissue from 40- and 80-day-old porcine fetuses was mechanically dissociated, and cells were cultured in serum-free F12/DMEM medium with B27 and N2 supplements, EGF and bFGF. In 3–5 days some cells divided and formed floating spheres that were dissociated to single cell suspension and formed secondary spheres in culture. At all time points tested, the spheres represented mixtures of undifferentiated cells stained with nestin and Ki-67 antibodies and already differentiated neurons (Tu-20, MAP2) and glia (GFAP). After being plated on laminin/fibronectin coated coverslips and cultured in medium containing 2% FBS or 1 μM retinoic acid, the spheres adhered to the surface, and flattened, and cells started to migrate out. After immunofluorescence staining with antibodies to neuronal markers Tu-20 and MAP2, glial marker GFAP and oligodendrocyte marker CNPase showed that all the three cell types were present among differentiated cells. The EpSC are characterized by a slow and unlimited proliferation rate and, therefore, they retain labelled precursors of DNA more extensively than other keratinocytes. The main pool of EpSC is located in the bulge region of the hair follicle root sheath. A new procedure to isolate porcine hair follicles including their root sheaths was developed. The keratinocytes that migrated from hair follicles in the presence of feeder cells were poorly differentiated and specifically expressed galectin-1 or galectin-1-binding sites in their nuclei in co-localization with ΔNp63α. The exclusion of feeder cells from experimental system induced formation of spheroid bodies from these keratinocytes. Approximately one-third of these spheroids were able to adhere to a surface precolonized with feeder cells and to start forming normally growing colonies. Porcine hair follicles represent an excellent model for study of the functional phenotype of hair follicle-originated keratinocytes, and the endogenous lectin Gal-1 seems to be a potential marker of the porcine stem cell compartment of the hair follicle under in vitro conditions.

Relationships between colony forming efficiency and parameters of intrinsic radiosensitivity

Purpose: In an attempt to determine whether radiosensitivity is correlated with colony forming efficiency (CFE), a large amount of data have been analysed from the literature. Materials and methods: The survival curves of 446 human cell lines irradiated in exponentially growing phase in vitro are included in this study. Technical factors such as culture type and the use of feeder cells were considered cofactors in addition to the genetic and histological origin of the cells. Intrinsic radiosensitivity is expressed in terms of the parameters of the linear quadratic model and the single-hit multitarget model. Results: It is shown that low CFE is characteristic of cells plated in agar and cells from primary biopsies. Cells plated in the presence of feeder cells have, in general, higher CFE than cells plated without feeder cells. A positive correlation is observed between intrinsic radiosensitivity and CFE: the higher the CFE, the more resistant the cell line. This relationship is particularly obvious when radiosensitivity is expressed in terms of alpha, S 2 or D, parameters which essentially characterize the initial part of the survival curve. The correlation is also found within histological or genetic groups of cell lines. However, for a given cell line, there is no relationship between CFE and radiosensitivity among different experiments. Cells irradiated in the presence of feeder cells are less subject to this behaviour. Conclusions: CFE as well as radiosensitivity are intrinsic properties of a cell line. Experimental conditions determine the quality of the correlation between radiosensitivity and CFE.