Vitronectin production by human yolk sac carcinoma cells resembling parietal endoderm

Normal mesenchymal cells, normal epithelial cells and many transformed epithelial cells require serum attachment factors and extracellular matrix proteins for growth and differentiation in vitro, and recent evidence strongly supports a role for extracellular matrix molecules in the regulation of cell movement in vivo during early embryogenesis. We previously described the isolation and characterization of cell lines representative of three types of stem cells most commonly found in human adult testicular teratomas, namely embryonal carcinoma cells, yolk sac carcinoma cells resembling visceral endoderm and yolk sac carcinoma cells resembling parietal endoderm (endodermal sinus tumour cells). Of these three cell types, only endodermal sinus tumour cells, which show particularly malignant behaviour in vivo, have no serum requirement for attachment and growth in vitro. Supernatants from endodermal sinus tumour cells support the attachment of embryonal carcinoma cells in serum-free medium. We demonstrate here that endodermal sinus tumour cells, but not other cell types isolated from testicular teratomas, secrete the serum attachment protein, vitronectin (also known as serum-spreading factor, S-protein or epibolin), as well as fibronectin, laminin and type IV collagen, into serum-free medium. Purified vitronectin from medium conditioned by endodermal sinus tumour cells supported both attachment and spreading of embryonal carcinoma cells in vitro, whereas cells attached but did not spread properly on surfaces coated with fibronectin or laminin. Peptides containing the RGD cell recognition sequence common to many attachment proteins blocked attachment of endodermal sinus tumour cells to untreated tissue-culture plastic in serum-free medium. The results suggest a possible role for vitronectin in regulating cell motility and growth in early development, and in the invasion and spread of teratomas in vivo.     

Clinically translatable cell tracking and quantification by MRI in cartilage repair using superparamagnetic iron oxides

BACKGROUND: Articular cartilage has very limited intrinsic regenerative capacity, making cell-based therapy a tempting approach for cartilage repair. Cell tracking can be a major step towards unraveling and improving the repair process of these therapies. We studied superparamagnetic iron oxides (SPIO) for labeling human bone marrow-derived mesenchymal stem cells (hBMSCs) regarding effectivity, cell viability, long term metabolic cell activity, chondrogenic differentiation and hBMSC secretion profile. We additionally examined the capacity of synovial cells to endocytose SPIO from dead, labeled cells, together with the use of magnetic resonance imaging (MRI) for intra-articular visualization and quantification of SPIO labeled cells. METHODOLOGY/PRINICIPAL FINDINGS: Efficacy and various safety aspects of SPIO cell labeling were determined using appropriate assays. Synovial SPIO re-uptake was investigated in vitro by co-labeling cells with SPIO and green fluorescent protein (GFP). MRI experiments were performed on a clinical 3.0T MRI scanner. Two cell-based cartilage repair techniques were mimicked for evaluating MRI traceability of labeled cells: intra-articular cell injection and cell implantation in cartilage defects. Cells were applied ex vivo or in vitro in an intra-articular environment and immediately scanned. SPIO labeling was effective and did not impair any of the studied safety aspects, including hBMSC secretion profile. SPIO from dead, labeled cells could be taken up by synovial cells. Both injected and implanted SPIO-labeled cells could accurately be visualized by MRI in a clinically relevant sized joint model using clinically applied cell doses. Finally, we quantified the amount of labeled cells seeded in cartilage defects using MR-based relaxometry. CONCLUSIONS: SPIO labeling appears to be safe without influencing cell behavior. SPIO labeled cells can be visualized in an intra-articular environment and quantified when seeded in cartilage defects.     

Isolation of colorectal cancer stem-like cells

This study is aimed at isolating colorectal cancer stem-like cells in vitro using a neurosphere assay method employed in isolating gliobastoma multiforme tumor cells. This was followed with confirmation of the isolated cells by flow cytometry, pluripotent genes expression and in vivo tumorigenicity assay. Using this culture assay, stem-like and non-stem-like CRC cells were isolated and expanded in vitro from purchased Balb/c mice induced with CT26 colorectal cancer (CRC) cell line. The procedure includes an initial mechanical dissociation and chemical digestion of tumor tissue and subsequently plating the resulting single cell suspension in serum-free medium (SFM) or serum-containing medium (SCM). This selectively permits growth of cancer stem-like cells in SFM and eliminates non-stem-like cancer cells through the process of anoikis or apoptosis. CRC stem cells derived cultures proliferated as non-adherent spheres in vitro in different shapes and sizes. These cells expressed cell surface markers previously reported for tumor stem cells, including CD44, CD133, CD166 and CD26 and formed tumors when implanted in severe combined immunodeficient mice in a concentration dependent manner. Importantly, the stem-like cells had self-renewal properties with significantly higher expression of the pluripotent stem cell genes NANOG, OCT4, and SOX2 compared to the adherent non-stem cells. Collectively, the results of this study indicate that SFM is a defined culture medium that enriches for CRC stem-like cells and represents a suitable in vitro model for the study of CRC stem-like cells. This finding may be useful in developing therapeutic strategies aimed at eradicating the tumorigenic subpopulation within colorectal cancer.     

In vivo MR imaging of magnetically labeled human embryonic stem cells

INTRODUCTION: Human embryonic stem cells (hES) have emerged as a potentially new therapeutic approach for treatment of heart and other diseases applying the concept of regenerative medicine. A method for in vivo visualization and tracking of transplanted hES would increase our understanding of in vivo hES behavior in both experimental and clinical settings. The aim of this study was to evaluate the feasibility of magnetic labeling and visualization of hES with magnetic resonance imaging (MRI). METHODS: hES were established and expanded according to standard procedures. After expansion, the cells were cultured under feeder free conditions and magnetically labeled by addition of dextran-coated Ferrum-oxide particles (Endorem) to the medium. Accumulation of small particles of iron-oxide (SPIO) in hES was assessed by Prussian blue staining and electron microscopy. For in vitro MRI, the labeled and unlabeled hES were examined in cell solution and after transplantation into explanted mouse heart ( approximately 100,000 cells) on a Bruker Avance DMX 500 vertical magnet at 11.75 T. A multi-slice, multi spin-echo T(2)-weighted images were obtained. For in vivo imaging, the experiments were performed on male Sprague-Dawley using Bruker Biospec 2.35 T magnet. The hES were directly injected ( approximately 500,000 cells) after surgical procedure (thoracotomy) into anterior left ventricular (LV) wall. Multi-slice T(2)-weighted gradient echo images were obtained using cardiac gating. RESULTS: hES appeared to be unaffected by magnetic labeling and maintained their ability to proliferate and differentiate. No additive agent for membrane permeabilisation was needed for facilitation of intracellular SPIO accumulation. Prussian blue and electron microscopy have revealed numerous iron particles in the cytoplasm of hES. On T(2)-weighted images, the labeled cells have shown well-defined hyopintense areas at the site of injection in anterior LV wall both in vitro and in vivo. CONCLUSIONS: It is feasible to magnetically label and visualize hES both in vitro and in vivo. MR visualization of magnetically labeled hES may be a valuable tool for in vivo tracking of hES.     

MRI-based Visualization of Iron-labeled CD133+ Human Endothelial Progenitor Cells

The objective of this study is to build up a kind of effective approach to multiply CD133+ endothelial progenitor cells (EPCs) and visualize cells by labeling with two FDA-approved agents based on MRI technique. CD133+ cells were separated by immunomagnetic microbeads selection and grew with serum-free medium. Seven days later, CD133+ cell production was collected and co-incubated with iron complex for 24 h for labeling. The iron-labeled cells were suspended into agarose gel and scanned by MRI for visualization. Labeled cells were also analyzed for cell viability. Iron can be effectively introduced into CD133+ EPCs plasma in culture and visualized by changing the MRI signal intensity. Iron had no influence on cell viability. CONCLUSION: Iron substance can be applied to label CD133+ cells without cytotoxicity and iron-labeled cells can be visualized by MRI image. Due to the non-invasive property and repeatability of MRI technology and this kind of method could be used for tracing in vivo stem cells in the future.     

Practical methods for handling human periodontal ligament stem cells in serum-free and serum-containing culture conditions under hypoxia: implications for regenerative medicine

Stem cell-based therapies depend on the reliable expansion of patient-derived mesenchymal stem cells (MSCs) in vitro. The supplementation of cell culture media with serum is associated with several risks; accordingly, serum-free media are commercially available for cell culture. Furthermore, hypoxia is known to accelerate the expansion of MSCs. The present study aimed to characterize the properties of periodontal ligament-derived MSCs (PDLSCs) cultivated in serum-free and serum-containing media, under hypoxic and normoxic conditions. Cell growth, gene and protein expression, cytodifferentiation potential, genomic stability, cytotoxic response, and in vivo hard tissue generation of PDLSCs were examined. Our findings indicated that cultivation in serum-free medium does not affect the MSC phenotype or chromosomal stability of PDLSCs. PDLSCs expanded in serum-free medium exhibited more active growth than in fetal bovine serum-containing medium. We found that hypoxia does not alter the cell growth of PDLSCs under serum-free conditions, but inhibits their osteogenic and adipogenic cytodifferentiation while enabling maintenance of their multidifferentiation potential regardless of the presence of serum. PDLSCs expanded in serum-free medium were found to retain common MSC characteristics, including the capacity for hard tissue formation in vivo. However, PDLSCs cultured in serum-free culture conditions were more susceptible to damage following exposure to extrinsic cytotoxic stimuli than those cultured in medium supplemented with serum, suggesting that serum-free culture conditions do not exert protective effects against cytotoxicity on PDLSC cultures. The present work provides a comparative evaluation of cell culture in serum-free and serum-containing media, under hypoxic and normoxic conditions, for applications in regenerative medicine.     

Size and charge heterogeneity of murine IgG-binding factors (IgG-BF)

Size and charge of murine IgG-binding factors (IgG-BF) were determined. Four different sources were used to produce the factors: a) cells of a T cell hybrid (T2D4) constitutively secreting IgG-BF upon incubation in serum-free medium, b) T2D4 cells incubated with mouse monoclonal IgG1 antibody in order to induce in vitro the production of isotype-specific IgG1-BF, c) T2D4 cells induced in vivo by passage as ascites in nude mice and incubated in serum-free medium, and d) in vivo alloantigen-activated T cells (ATC) incubated in serum-free medium. IgG-BF were affinity purified on Sepharose beads coated with rabbit or mouse IgG and identified by their biologic activities, i.e., inhibition of in vitro secondary IgG antibody production to SRBC and inhibition of rosette formation between Fc gamma receptor-positive spleen cells and rabbit IgG-sensitized erythrocytes. IgG-BF produced by either of these cell sources was found to be heterogeneous in both size and charge. In each case, IgG-BF activities were recovered in three fractions of apparent Mr-74,000 to 78,000, 35,000 to 40,000, and 19,000 to 23,000-and in four fractions of pI-4.7 (or 5.3, depending on experimental conditions), 6.5, 7.7, and 8.4. Moreover, IgG-BF translated in vitro from T2D4 poly A RNA by using rabbit reticulocyte lysate exhibited the same heterogeneity. Thus, IgG-BF contain different proteins exerting similar biologic activities.     

Cells captured from spatium intermusculare by porous material exhibit the characteristics of stem cells

Most of the current methods to capture stem cells are very complicated. Our new discovery of acquiring adult stem cells by implanting three dimension (3-D) porous material into the spatium intermusculare of mice hind limbs would bring hope to achieve autologous stem cells transplantation. We discovered that a great number of cells migrated into the 3-D porous material implanted in vivo. Furthermore, the migrating cells exhibited stem cell properties (CD34(+), Sca-1(+), GFAP(+), alphafetoprotein(+)) and were hematogeous (CD45(+)) and CD105(+). The ability of migrating cells to undergo differentiation into hematopoietic lineages was tested with methylcellulose medium. These findings demonstrate that the cells captured from spatium intermusculare by implanting 3-D porous material exhibit the characteristics of stem cells.     

Epithelial-mesenchymal interactions allow for epidermal cells to display an in vivo-like phenotype in vitro

We here report that preservation of the basic epithelial-mesenchymal interactions allows for highly complex ex vivo function of epidermal cells. The approach taken is based on the preparation of organ fragments that preserve the basic epithelial/mesenchymal interactions but also ensure appropriate diffusion of nutrients and gases to all cells. Human and mice keratinocytes in such organ fragments, remain viable, proliferate and express epidermal-specific gene products when cultured in serum-free medium without added growth factors, for several weeks in vitro. When implanted into syngeneic animals they remain viable, become vascularized and continue to function and transcribe tissue-specific gene products for several months. Such fragments allow primary cells ex vivo to preserve most of the functional attributes of the in vivo system. Clearly, the effect of the extracellular matrix is critical in this system in order for the cells to proliferate and differentiate ex vivo. We are not aware of any other system which allows for localized expression of epidermal-specific genes ex vivo for significant periods in culture in defined serum-free medium.     

Routine culturing of normal,dysplastic and malignant human mammary epithelial cells from small tissue samples

Summary We compared the growth and morphology of normal, dysplastic and malignant human mammary epithelial cells (HMEC) in medium containing 5% human serum, a serum-free medium (32) and serum-free medium with a low Ca⁺⁺ concentration. Tissues were dissociated and epithelial organoids or single cells were seeded onto collagen-coated dishes. The cells grew in serum-containing medium, but growth of fibroblasts was also stimulated. The serum-free medium consistently selected for and stimulated the growth of epithelial cells. There was little advantage in reducing the Ca⁺⁺ concentration to further increase cell yield. This serum-free primary culture system allows us to routinely prouce sufficient numbers of HMEC from small tissue samples for molecular biological investigations. Furthermore, the maintenance of cells in a defined medium can provide a system for evaluating the direct effects of factors on gene expression. This work was supported by a grant from the National Cancer Institute of Canada and funds contributed by Mr. B. T. Wharton in memory of his wife, Nadia.     

Generation of multivirus-specific T cells by a single stimulation of peripheral blood mononuclear cells with a peptide mixture using serum-free medium

Background: Restoration of virus-specific immunity by virus specific T cells (VSTs) offers an attractive alternative to conventional drugs, and can be highly effective in immunocompromised patients, including hematopoietic stem cell transplant (HSCT) recipients. However, conventional VSTs manufacture requires preparation of specialized antigen-presenting cells (APCs), prolonged ex vivo culture in serum-containing medium and antigen re-stimulation with viruses or viral vectors to provide viral antigens for presentation on APCs. Methods: To simplify this complex process, we developed a method to generate multiple VSTs by direct stimulation of peripheral blood mononuclear cells (PBMCs) with overlapping peptide libraries in serum-free medium. Results: We generated VSTs that targeted seven viruses (cytomegalovirus [CMV], Epstein-Barr virus [EBV], adenovirus [AdV], human herpesvirus 6 [HHV-6], BK virus [BKV], JC virus [JCV] and Varicella Zoster virus [VZV]) in a single line. The phenotype, growth and specificity of multiple VSTs produced in serum-free medium were equivalent to those generated in conventional serum-containing medium. Discussion: The use of serum-free medium allows this approach to be readily introduced to clinical practice with lower cost, greater reproducibility due to the absence of batch-to-batch variability in serum and without concerns for infectious agents in the serum used. This simplified approach will now be tested in recipients of Human Leukocyte Antigen (HLA)–matched sibling HSCT.     

Serum-free ex vivo expansion of CD34(+) hematopoietic progenitor cells

In an effort to obtain defined culture conditions for ex vivo expansion of hematopoietic stem and progenitor cells which avoid the supplementation of serum, we cultured human CD34(+) hematopoietic progenitor cells in a chemically defined, serum-free medium in the presence of hematopoietic growth factors (HGFs), stem cell factor (SCF), interleukin (IL)-1beta, IL-3, IL-6, and erythropoietin (EPO). A medium, SFM-1, was prepared according to a protocol previously optimized for semisolid progenitor cell assays containing Iscove's Modified Dulbecco's Medium (IMDM) plus cholesterol, bovine serum albumin, transferrin, nucleotides and nucleosides, insulin, and beta-mercaptoethanol. In static cultures seeded with CD34(+)-enriched progenitor cells isolated from human peripheral blood, a mean 76.6-fold expansion of total nucleated cells and a mean 4.6-fold expansion of colony-forming cells (CFC) was recorded after 14 days. Morphological analysis of the expanded cells revealed formation of myeloid, erythroid, and megakaryocytic cells. Flow cytometric analysis indicated that CD34(+) antigen expressing cells were maintained to a limited degree only, and cell populations expressing surface markers for myeloid (CD33, CD14, and CD15) and megakaryocytic (CD41a) lineages predominated. Within SFM-1, bovine serum albumin (BSA), cholesterin, and transferrin represented the most critical components needed for efficient total cell and CFC expansion. Addition of autologous patient plasma (APP) or fetal calf serum (FCS) to SFM-1 resulted in inferior cell amplification and CFC formation compared to controls in SFM-1, indicating that the components used in SFM-1 could replace exogenous serum. Four commercially available serum-free media resulted in either comparable or lower total cell and CFC yields as SFM-1. The transplantation potential of CD34(+) cells after culture in SFM-1 was assayed using limiting dilution analysis on preformed irradiated bone marrow stroma and revealed maintenance of long-term bone marrow culture initiating cell (LTCIC) levels during the culture period. These data indicate that HGF-supported multilineage ex vivo expansion of human CD34(+) hematopoietic progenitor cells is feasible using an IMDM-based culture medium which contains a restricted number of additives, resulting in analogous or improved yields of both primitive and differentiated cells compared to previously established protocols. We suggest that this culture protocol is of advantage when working with pharmaceutical-grade preparations under serum-free conditions.     

Ferritin as a reporter gene for in vivo tracking of stem cells by 1.5-T cardiac MRI in a rat model of myocardial infarction

The methods currently utilized to track stem cells by cardiac MRI are affected by important limitations, and new solutions are needed. We tested human ferritin heavy chain (hFTH) as a reporter gene for in vivo tracking of stem cells by cardiac MRI. Swine cardiac stem/progenitor cells were transduced with a lentiviral vector to overexpress hFTH and cultured to obtain cardiospheres (Cs). Myocardial infarction was induced in rats, and, after 45 min, the animals were subjected to intramyocardial injection of ～200 hFTH-Cs or nontransduced Cs or saline solution in the border zone. By employing clinical standard 1.5-Tesla MRI scanner and a multiecho T2* gradient echo sequence, we localized iron-accumulating tissue only in hearts treated with hFTH-Cs. This signal was detectable at 1 wk after infarction, and its size did not change significantly after 4 wk (6.33 ± 3.05 vs. 4.41 ± 4.38 mm(2)). Cs transduction did not affect their cardioreparative potential, as indicated by the significantly better preserved left ventricular global and regional function and the 36% reduction in infarct size in both groups that received Cs compared with control infarcts. Prussian blue staining confirmed the presence of differentiated, iron-accumulating cells containing mitochondria of porcine origin. Cs-derived cells displayed CD31, α-smooth muscle, and α-sarcomeric actin antigens, indicating that the differentiation into endothelial, smooth muscle and cardiac muscle lineage was not affected by ferritin overexpression. In conclusion, hFTH can be used as a MRI reporter gene to track dividing/differentiating stem cells in the beating heart, while simultaneously monitoring cardiac morpho-functional changes.     

Magnetodendrimers allow endosomal magnetic labeling and in vivo tracking of stem cells.

Magnetic resonance (MR) tracking of magnetically labeled stem and progenitor cells is an emerging technology, leading to an urgent need for magnetic probes that can make cells highly magnetic during their normal expansion in culture. We have developed magnetodendrimers as a versatile class of magnetic tags that can efficiently label mammalian cells, including human neural stem cells (NSCs) and mesenchymal stem cells (MSCs), through a nonspecific membrane adsorption process with subsequent intracellular (non-nuclear) localization in endosomes. The superparamagnetic iron oxide nanocomposites have been optimized to exhibit superior magnetic properties and to induce sufficient MR cell contrast at incubated doses as low as 1 microg iron/ml culture medium. When containing between 9 and 14 pg iron/cell, labeled cells exhibit an ex vivo nuclear magnetic resonance (NMR) relaxation rate (1/T2) as high as 24-39 s-1/mM iron. Labeled cells are unaffected in their viability and proliferating capacity, and labeled human NSCs differentiate normally into neurons. Furthermore, we show here that NSC-derived (and LacZ-transfected), magnetically labeled oligodendroglial progenitors can be readily detected in vivo at least as long as six weeks after transplantation, with an excellent correlation between the obtained MR contrast and staining for beta-galactosidase expression. The availability of magnetodendrimers opens up the possibility of MR tracking of a wide variety of (stem) cell transplants.     

Properties of a novel magnetized alginate for magnetic resonance imaging

Implanting recombinant cells encapsulated in alginate microcapsules to secrete therapeutic proteins has been proven clinically effective in treating several murine models of human diseases. However, once implanted, these microcapsules cannot be assessed without invasive surgery. We now report the preparation and characterization of a novel ferrofluid to render these microcapsules visible with magnetic resonance imaging (MRI). The ferrofluid was prepared as a colloidal iron oxide stabilized in water by alginate. The presence of iron particles in the ferrofluid was verified with chemical titration, dynamic light scattering, and magnetization measurement. The microcapsules fabricated with various concentrations of the ferrofluid in the core, or on the surface of alginate microcapsules, or both, all produced microcapsules with smooth surfaces as shown with light and scanning electron microscopy. However, at the nanoscale level, as revealed with atomic force microscopy, the ferrofluid-fabricated microcapsules demonstrated increased granularity, particularly when the ferrofluid was used to laminate the surface. From the force spectroscopy measurements, these modified microcapsules showed increasing surface rigidity in the following order: traditional alginate < ferrofluid in the core < ferrofluid on the surface. Although the mechanical stability of low-concentration ferrofluid (0.1%) microcapsules was reduced, increasing concentrations, up to 20%, were able to improve stability. When these ferrofluid microcapsules were examined with MRI, their T(2) relaxation time was reduced, thereby producing increased contrast readily detectable with MRI, whereas the traditional alginate microcapsules showed no difference when compared with water. In conclusion, such ferrofluid-enhanced alginate is suitable for fabricating microcapsules that offer the potential for in vivo tracking of implanted microcapsules without invasive surgery.     

Messenger RNA electroporation of human monocytes,followed by rapid in vitro differentiation,leads to highly stimulatory antigen-loaded mature dendritic cells

Dendritic cells (DC) are professional Ag-capturing and -presenting cells of the immune system. Because of their exceptional capability of activating tumor-specific T cells, cancer vaccination research is now shifting toward the formulation of a clinical human DC vaccine. We developed a short term and serum-free culture protocol for rapid generation of fully mature, viable, and highly stimulatory CD83(+) DC. Human monocytes were cultured for 24 h in serum-free AIM-V medium, followed by 24-h maturation by polyriboinosinic polyribocytidylic acid (polyI:C). Short term cultured, polyI:C-maturated DC, far more than immature DC, showed typical mature DC markers and high allogeneic stimulatory capacity and had high autologous stimulatory capacity in an influenza model system using peptide-pulsed DC. Electroporation of mRNA as an Ag-loading strategy in these cells was optimized using mRNA encoding the enhanced green fluorescent protein (EGFP). Monocytes electroporated with EGFP mRNA, followed by short term, serum-free differentiation to mature DC, had a phenotype of DC, and all showed positive EGFP fluorescence. Influenza matrix protein mRNA-electroporated monocytes cultured serum-free and maturated with polyI:C showed high stimulatory capacity in autologous T cell activation experiments. In conclusion, the present short term and serum-free ex vivo DC culture protocol in combination with mRNA electroporation at the monocyte stage imply an important reduction in time and consumables for preparation of Ag-loaded mature DC compared with classical DC culture protocols and might find application in clinical immunotherapy settings.     

The Activin A-Dependent Proliferation of PCC3/A/1 Embryonal Carcinoma Cells in Serum-Free Medium

Examination of the growth requirements of murine embryonal carcinoma cells (EC cells) or embryonic stem cells (ES cells) in serum-free medium revealed that PCC3 EC cells required activin A to grow and/or survive in such medium. In the absence of activin A, PCC3 cells began to disintegrate within 3 days under any serum-free conditions examined. P19 and AT805 EC cells grew even in serum-free medium without activin A but their growth rates were slightly facilitated by its addition. F9 EC cells also grew in the medium without activin A and its addition somewhat inhibited their growth rate. Three independently isolated ES cell lines and feeder-dependent PSA-1 EC cells also grew in serum-free medium without activin A if leukemia inhibitory factor (LIF) was supplemented. The addition of activin A had little effect on their growth rates. These findings suggest that PCC3 EC cells are a sort of nutritional mutant requiring activin A, thus making them useful in stidies on the growth regulatory mechanisms of EC/ES cells and/or the action of activin on EC/ES cells.     

Effects of growth factors on the differentiation of murine ESC into type II pneumocytes

We have previously shown that embryonic stem cells (ESC) can be directed to differentiate into alveolar type II cells by provision of a serum-free medium designed for in vitro maintenance of mature alveolar epithelial cells (small airway growth medium: SAGM), although the target cell yield was low. SAGM comprises a basal serum-free medium (SABM) plus a series of defined supplements. In order to try increase the proportion of pneumocytes in differentiated cultures, we aimed in this study to determine the effects on murine ESC of each of the individual growth factors in SAGM. In accordance with our previous reports, expression of surfactant protein C (SPC) and its mRNA was used to monitor differentiation of type II pneumocytes. Surprisingly, we found that addition of each factor separately to SABM decreased the expression of SPC mRNA when compared with the effect of SABM alone. Thus, it seems that the observed enhancement by SAGM of pneumocyte differentiation from murine ESC can, in fact, be attributed to the provision of a serum-free environment.     

Human erythroid cells produced ex vivo at large scale differentiate into red blood cells in vivo

New sources of red blood cells (RBCs) would improve the transfusion capacity of blood centers. Our objective was to generate cells for transfusion by inducing a massive proliferation of hematopoietic stem and progenitor cells, followed by terminal erythroid differentiation. We describe here a procedure for amplifying hematopoietic stem cells (HSCs) from human cord blood (CB) by the sequential application of specific combinations of growth factors in a serum-free culture medium. The procedure allowed the ex vivo expansion of CD34+ progenitor and stem cells into a pure erythroid precursor population. When injected into nonobese diabetic, severe combined immunodeficient (NOD/SCID) mice, the erythroid cells were capable of proliferation and terminal differentiation into mature enucleated RBCs. The approach may eventually be useful in clinical transfusion applications.     

Characterization of a serum-free culture system comparing growth factor requirements of transformed and untransformed cells

We describe the first completely serum-free model culture system for comparing growth control in transformed and untransformed cells. Continuous maintenance of untransformed AKR-2B fibroblasts and chemically transformed AKR-MCA cells in the presence of serum-free medium containing epidermal growth factor (E), insulin (I), and transferrin (T) resulted in cell lines which proliferated with similar doubling times (14 h), comparable to parental lines maintained in 10% serum (16 h). The transformed MCA-SF cells and untransformed AKR-SF cells did not differ in their saturation densities in medium containing E + I + T. However, the monolayer proliferation of MCA-SF cells was significantly greater than that of the AKR-SF cells in the presence of E + T, I + T, or T alone. Both cell lines required T to proliferate in monolayer culture. [3H]-Thymidine incorporation experiments and autoradiographic analysis indicated that quiescent MCA-SF cells could reenter the cell cycle by addition of nutrients alone. The combination of E + I + T produced no additional stimulation of DNA synthesis. In contrast, individual polypeptide growth factors (E, I, IGF-I, PDGF, FGF a or b, or TGF-beta 1) were required to elicit a mitogenic response in the untransformed AKR-SF cells. Peak mitogenesis occurred from 18-20 h for all growth factors except TGF-beta 1 (32 h). Neither AKR-SF nor MCA-SF cells could grow with anchorage independence in serum-free medium, unless both TGF-beta 1 and FGF a or b were simultaneously present. The results indicate that this well-defined, serum-free model system can be utilized to detect growth factor-related alterations associated with the transformed state.