Spheroids In Serum-free Medium Research Articles

Abstract 36: Comparing twenty-two matched patient-derived cell lines developed from either patient, PDX, or organoid tumor cell material

Abstract The National Cancer Institute has developed a Patient-Derived Models Repository (NCI PDMR; https://pdmr.cancer.gov) comprised of quality-controlled, early passage, and clinically-annotated patient-derived tumor xenografts (PDXs), organoids (PDOrgs), cell cultures (PDCs), and cancer associated fibroblasts (CAFs) available with genomic data to the extramural community for research use. Models are developed by the NCI PDMR in decreasing order of complexity, specifically 1) patient material can be used to develop PDXs, PDOrgs, PDCs, and CAFs, 2) PDX material for PDOrgs and PDCs, and 3) PDOrg material for PDCs, but a PDC is never used to develop a PDOrg or PDX. Eleven pairs of 22 matched PDCs have been developed in parallel from either patient, PDX, patient-derived organoid, or PDX-derived organoid tumor material and sequenced by WES and RNASeq. Genetic stability was assessed using multiple approaches including microsatellite instability (MSI) generated from MSISensor2, percentage of genomic loss of heterozygosity (LOH) using a set of ~800k heterozygous SNPs from a population level genomic database (gnomAD), pairwise Spearman correlation based on BIN level copy number (CN)/RNA expression profiles, and OncoKB annotated oncogenic/likely oncogenic variants. No systematic differences were observed within PDC pairs derived from different origins or compared to their patient and/or PDX material in MSI, LOH% and RNA expression profile but pairwise Spearman correlation (0.66-0.88) in CNV profiles were somewhat variable, likely due to low sequencing depth. In one PDC pair (299254), 3 out of 12 OncoKB annotated Indels and CNV showed opposite level of variant allele frequencies/CN when comparing a model derived from patient material to one developed from a PDX-derived organoid, possibly driven by a lineage-specific subclonal outgrowth when compared to patient and PDX data. Phenotypic characteristics of matched PDCs also overall show no major differences, though variability in growth rates and the ability to form spheroids in serum-free medium were noted. In one pair (919269), the PDC derived from patient material was able to form a cell line xenograft (CLX) in NSG mice but not the PDC developed from a patient-derived organoid. Overall, these models demonstrate a high degree of concordance at the genetic and phenotypic level when compared to the originating patient and/or PDX tumor. Though further characterization (e.g., preclinical drug testing) may be needed to define differences between matched PDC pairs, lack of access to patient tissue or failure to generate tumor cell cultures from one source of material should not hamper development of preclinical in vitro models from other patient-derived model types as long as the source-of-origin is clearly defined. Funded by NCI Contract No. HHSN261200800001E Citation Format: Cindy R. Timme, Ting-Chia Chang, Sergio Y. Alcoser, Gareth Bliss, Carrie Bonomi, Suzanne Borgel, John Carter, Alice Chen, Li Chen, Kevin Cooper, Biswajit Das, Kelly Dougherty, Lindsay Dutko, Marion Gibson, Michelle M. Gottholm-Ahalt, Tara Grinnage-Pulley, Shahanawaz Jiwani, Keegan Kalmbach, Chris Karlovich, Kimberly Klarmann, Tiffanie Chase, Michael Mullendore, Matthew Murphy, Kevin Plater, Gloryvee Rivera, Jessica Steed, Luke Stockwin, Yvonne A. Evrard, Mickey Williams, Dianne L. Newton, Melinda G. Hollingshead, James H. Doroshow. Comparing twenty-two matched patient-derived cell lines developed from either patient, PDX, or organoid tumor cell material [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 36.

Optimizing Conditions for Spheroid Formation of Dental Pulp Cells in Cell Culture.

Spheroid formation is a well-known feature of stem/progenitor cells. Dental pulp cells (DPCs) cultured in serum-free medium can also form spheroids. However, the success rate varies largely depending on various factors. This study aimed to explore these factors and optimize the conditions. Primary DPCs were obtained from 6 wisdom teeth. Possible influencing factors including donor teeth, concentrations of the KnockOut Serum Replacement (KSR), seeding density (regarding surface and volume), passage and freezing were tested. DPCs from all 6 donor teeth formed spheroids in serum-free medium. Number, size, and total area of spheroids varied among different donor teeth. Optimal concentration of the KSR and seeding densities also varied from tooth to tooth. Generally, high KSR and high cell density lead to better spheroid formation. However, very high KSR and cell density can also lead to cell death and the fusion of spheroids to irregular aggregates. An initial setting can be recommended as: Serum-free MEM plus 10-15% KSR and seeding densities of 1-2×105 cells/ml and 2×105 cells/cm2 These parameters provide a direction for optimizing the condition for obtaining spheroids from human DPCs.

Rapid and Efficient Production of Human Functional Mast Cells through a Three-Dimensional Culture of Adipose Tissue-Derived Stromal Vascular Cells.

Mast cells (MC) are innate immune cells involved in many physiological and pathological processes. However, studies of MC function and biology are hampered by the difficulties to obtain human primary MC. To solve this problem, we established a new method to produce easily and rapidly high numbers of MC for in vitro studies using human adipose tissue, which is an abundant and easy access tissue. Stromal vascular fraction of adipose tissue, obtained from human abdominal dermolipectomy, was cultured as spheroids in serum free medium supplemented in stem cell factor. Using this method, we generated, within 3 wk, a highly pure population of connective tissue-type MC expressing MC typical peptidases (tryptase, chymase, and carboxypeptidase-A3) with a yield increasing over time. Stem cell factor was required for this culture, but unlike MC derived from CD34+ cells, this culture did not depend on IL-3 and -6. MC obtained with this method degranulated following FcεRI cross-linking or stimulation by C5a, compound 48/80, and substance P. Interestingly, activation by anti-IgE of both white adipose tissue-MC and MC obtained from peripheral blood-derived CD34+ pluripotent progenitor cells induced the production of PGs as well as proinflammatory cytokines (TNF-α, Il-6, and GM-CSF). In conclusion, we developed a new time saving and reproducible method to produce highly pure and functional human MC in 3 wk from human adipose tissue.

Phenethyl isothiocyanate inhibits colorectal cancer stem cells by suppressing Wnt/β-catenin pathway.

Cancer stem cells (CSCs) are considered to play essential roles in the process of origination, proliferation, migration, and invasion of cancer, and their properties are regulated by Wnt/β-catenin pathway. Phenethyl isothiocyanate (PEITC) is a natural product obtained from cruciferous vegetables with anticancer activities. The present study aimed to investigate the inhibitory effect and the underlying mechanisms of PEITC on colorectal CSCs. In this study, we found that PEITC can significantly reduce the size and number of colorectal cancer cell spheroids in serum-free medium. With increasing PEITC concentrations (10-40μM), the number of spheroids was reduced to about 10% of the control group, and the percentage of CD133+ cells was decreased by about 3-16 folds. PEITC also decreased the expression of CSC markers. Meanwhile, inhibition of proliferation as well as induction of apoptosis of colorectal CSCs was observed after PEITC treatment. Furthermore, through activating Wnt/β-catenin pathway with LiCl, the inhibitory effects of PEITC on colorectal CSCs were diminished. Our data suggested that PEITC can be an effective inhibitor of colorectal CSCs by targeting Wnt/β-catenin pathway.

Evaluation of the effect of hyperthermia and electron radiation on prostate cancer stem cells

The aim of this study was to investigate the effect of hyperthermia, 6MeV electron radiation and combination of these treatments on cancer cell line DU145 in both monolayer culture and spheroids enriched for prostate cancer stem cells (CSCs). Flowcytometric analysis of the expression of molecular markers CD133+/CD44+ was carried out to determine the prostate CSCs in cell line DU145 grown as spheroids in serum-free medium. Following monolayer and spheroid culture, DU145 cells were treated with different doses of hyperthermia, electron beam and combination of them. The survival and self-renewing of the cells were evaluated by colony formation assay (CFA) and spheroid formation assay (SFA). Flowcytometry results indicated that the percentage of CD133+/CD44+ cells in spheroid culture was 13.9-fold higher than in the monolayer culture. The SFA showed significant difference between monolayer and spheroid culture for radiation treatment (6Gy) and hyperthermia (60 and 90min). The CFA showed significantly enhanced radiosensitivity in DU145 cells grown as monolayer as compared to spheroids, but no effect of hyperthermia. In contrast, for the combination of radiation and hyperthermia the results of CFA and SFA showed a reduced survival fraction in both cultures, with larger effects in monolayer than in spheroid culture. Thus, hyperthermia may be a promising approach in prostate cancer treatment that enhances the cytotoxic effect of electron radiation. Furthermore, determination and characterization of radioresistance and thermoresistance of CSCs in the prostate tumor is the key to develop more efficient therapeutic strategies.

Ecto-5'-nucleotidase (CD73) is a biomarker for clear cell renal carcinoma stem-like cells.

Identification of a specific biomarker for cancer stem cells (CSCs) is of potential applications in the development of effective therapeutic strategies for renal cell carcinoma (RCC). In this study, both the RCC cell line 786-O and surgically removed clear cell RCC (ccRCC) tissues were implemented to grew as spheroids in serum-free medium supplemented with mitogens. This subpopulation possessed key characteristics defining CSCs. We also identified that surgically removed ccRCC tissues were heterogenic and there was a subpopulation of cells that was highly stained with rhodamine-123. Based on membrane-proteomic analyses, CD73 was identified as a candidate biomarker. We further found that CD73high cells were highly tumorigenic. As few as 100 CD73high cells were capable of forming xenograft tumors in non obese diabetic/severe combined immunodeficiency disease mice, whereas 1 × 105 CD73low cells did not initiate tumor formation. During successive culture, the CD73high population regenerated both CD73high and CD73low cells, whereas the CD73low population remained low expression level of CD73. Furthermore, the CD73high cells were more resistant to radiation and DNA-damaging agents than the CD73low cells, and expressed a panel of ‘stemness’ genes at a higher level than the CD73low cells. These findings suggest that a high level of CD73 expression is a bona fide biomarker of ccRCC stem-like cells. Future research will aim at the elucidation of the underlying mechanisms of CD73 in RCC development and the distinct aspects of ccRCC stem-like cells from other tumor types.

Three-dimensional spheroid culture of human umbilical cord mesenchymal stem cells promotes cell yield and stemness maintenance.

Mesenchymal stem cell (MSC) transplantation is a promising treatment of many diseases. However, conventional techniques with cells being cultured as a monolayer result in slow cell proliferation and insufficient yield to meet clinical demands. Three-dimensional (3D) culture systems are gaining attention with regard to recreating a complex microenvironment and to understanding the conditions experienced by cells. Our aim is to establish a novel 3D system for the culture of human umbilical cord MSCs (hUC-MSCs) within a real 3D microenvironment but with no digestion or passaging. Primary hUC-MSCs were isolated and grown in serum-free medium (SFM) on a suspension Rocker system. Cell characteristics including proliferation, phenotype and multipotency were recorded. The therapeutic effects of 3D-cultured hUC-MSCs on carbon tetrachloride (CCl4)-induced acute liver failure in mouse models were examined. In the 3D Rocker system, hUC-MSCs formed spheroids in SFM and maintained high viability and active proliferation. Compared with monolayer culture, the 3D-culture system yielded more hUC-MSCs cells within the same volume. The spheroids expressed higher levels of stem cell markers and displayed stronger multipotency. After transplantation into mouse, 3D hUC-MSCs significantly promoted the secretion of interferon-γ and interleukin-6 but inhibited that of tumor necrosis factor-α, thereby alleviating liver necrosis and promoting regeneration following CCl4 injury. The 3D culture of hUC-MSCs thus promotes cell yield and stemness maintenance and represents a promising strategy for hUC-MSCs expansion on an industrial scale with great potential for cell therapy and biotechnology.

Characterization of human dental pulp cells-derived spheroids in serum-free medium: Stem cells in the core

Spheroid models have led to an increased understanding of differentiation, tissue organization and homeostasis. In the present study, we have observed that under a serum-free medium, human dental pulp cells (DPCs) spontaneously formed spheroids, and could survive over 15 weeks. To characterize these spheroids, we investigated their dynamics, microenvironment, cell distribution, molecular profiles, and neuronal/osteogenic potential. Cell tracking assay showed that cells inside the spheroids have very slow cycling. Although the spheroids had hypoxia microenvironments, there were not any massive cell die-offs even after long-term cultivation. Whole mount immunofluorescence staining and histological analysis showed a distribution of stem cells in the central/intermediate zones of spheroids. qRT-PCR analysis demonstrated that the expression of stemness markers NANOG, TP63, and CD44 in the spheroids were much higher than within the monolayer cultures. Gene expression levels of neural markers CDH2, NFM, TUBB3, and CD24 in the spheroids were much higher than the monolayer DPCs and increased in a culture time-dependent manner. Without any neural induction, spheroid-derived cells spontaneously converted into neuron-like cells with positive staining of neural markers HuC/D and P75 under the serum-free medium for about 2 weeks. When the spheroids were transferred into osteogenic medium, they rapidly differentiated into osteo/odontogenic cells, especially the central original cells. Compared to the monolayer DPCs, mineralization in spheroids were significantly increased. This spheroid model offers a study tool to explore the molecular bases of stem cell homeostasis and tissue organization, and can be wildly used for nerve tissue and bone regeneration.

NANOG modulates stemness in human colorectal cancer

NANOG is a stem cell transcription factor that is essential for embryonic development, reprogramming normal adult cells and malignant transformation and progression. The nearly identical retrogene NANOGP8 is expressed in multiple cancers, but generally not in normal tissues and its function is not well defined. Our postulate is that NANOGP8 directly modulates the stemness of individual human colorectal carcinoma (CRC) cells. Stemness was measured in vitro as the spherogenicity of single CRC cells in serum-free medium and the size of the side population (SP) and in vivo as tumorigenicity and experimental metastatic potential in NOD/SCID mice. We found that 80% of clinical liver metastases express a NANOG with 75% of the positive metastases containing NANOGP8 transcripts. In all, 3-62% of single cells within six CRC lines form spheroids in serum-free medium in suspension. NANOGP8 is translated into protein. The relative expression of a NANOG gene increased 8- to 122-fold during spheroid formation, more than the increase in OCT4 or SOX2 transcripts with NANOGP8 the more prevalent family member. Short hairpin RNA (shRNA) to NANOG not only inhibits spherogenicity but also reduces expression of OCT4 and SOX2, the size of the SP and tumor growth in vivo. Inhibition of NANOG gene expression is associated with inhibition of proliferation and decreased phosphorylation of G2-related cell-cycle proteins. Overexpression of NANOGP8 rescues single-cell spherogenicity when NANOG gene expression is inhibited and increases the SP in CRC. Thus, NANOGP8 can substitute for NANOG in directly promoting stemness in CRC.

211 ISOLATION, PROPAGATION, AND CHARACTERIZATION OF PROSTATE CANCER STEM CELLS FROM PRIMARY TUMORS

You have accessJournal of UrologyStem Cell Research1 Apr 2012211 ISOLATION, PROPAGATION, AND CHARACTERIZATION OF PROSTATE CANCER STEM CELLS FROM PRIMARY TUMORS Rosa Park, Edison Zuniga, Rahuldev Bhalla, and Galina Botchkina Rosa ParkRosa Park Stony Brook, NY More articles by this author , Edison ZunigaEdison Zuniga Stony Brook, NY More articles by this author , Rahuldev BhallaRahuldev Bhalla Stony Brook, NY More articles by this author , and Galina BotchkinaGalina Botchkina Stony Brook, NY More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2012.02.264AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Survival for localized prostate cancer is high with surgery and radiation therapy. However, survival rates with metastatic prostate cancer remains low even with anti-androgen treatment. This limited effectiveness of standard anti-cancer therapies is attributed to the presence of prostate cancer stem cells (CSCs), which may be responsible for tumor initiation, development, invasion and response to treatment. Our goal is to identify and characterize an in vitro prostate cancer stem cell model for the evaluation of prostate CSC targeted drug effects. METHODS Samples of prostate cancer tissue were collected from 16 patients after radical prostatectomy. The harvested tissues were dissociated by enzymatic digestion in serum-free RPMI medium containg collagenases type II and type IV (Sigma-Aldrich). The dissociated cells were plated on both collagen I coated plates and nonadherent plates to grow single cell suspensions and 3D spheroids in serum free medium for propagation. A portion of these cells was injected into NOD/SCID to grow tumor xenografts. The cells were sorted and characterized under FACS and MACS analysis using markers CD133, CD44, CD166, CXCR and EpCAM. Other immunofluorescence staining and PCR arrays were conducted to further characterize cells. RESULTS 1 of 16 patient samples showed high tumorigenic capacity. The patient derived cells showed high expression of CD133, CXCR4, vimentin, and nestin. They demonstrated significantly higher tumor initiating capacity after transplantation into NOD/SCID mice with rapid tumor growth, and highest sphere-forming and clonogenic capacities compared to other cell phenotypes. Proteomics study of CSC enriched cells showed 51 up-regulated and 33 down-regulated proteins, many of which are involved in stem cell function, invasion, and metastasis, like vimentin and nestin. CONCLUSIONS Isolation of prostate cancer stem cells from primary tumors is difficult. However, we were able to demonstrate “stemness” by studying its morphology and proteonomic characteristics. Currently in vitro studies and in vivo studies of prostate CSCs using Docetaxel and SB-T-1214, developed by Stony Brook, are underway. Further efforts are needed to make patient derived stem cell lines in order to discover new CSC targeted therapies for androgen-independent prostate cancer. © 2012 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 187Issue 4SApril 2012Page: e88 Advertisement Copyright & Permissions© 2012 by American Urological Association Education and Research, Inc.MetricsAuthor Information Rosa Park Stony Brook, NY More articles by this author Edison Zuniga Stony Brook, NY More articles by this author Rahuldev Bhalla Stony Brook, NY More articles by this author Galina Botchkina Stony Brook, NY More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Isolation and identification of cancer stem cells from primary human ovarian cancer tissues

To isolate and identify the cancer stem cells from primary human ovarian cancer tissues. Fresh tumor tissues from five cases of pathologically diagnosed ovarian cancers were taken, minced and then digested with collagenase and hyaluronidase to obtain single cell suspension. The erythrocytes were removed with ACK Lysis buffer. The suspensions were sorted by magnetic activated cell sorting (MACS) using CD133-binding microbeads. Then the sorted CD133(+) cells were verified by flow cytometry. The cells were cultured in serum-free medium supplemented with EGF, bFGF, insulin and BSA, and grew into spheroids. Immunofluorescence, differentiation and tumor formation tests of the cells were performed to characterize the properties of cancer stem cells. The ovarian cancer stem cells were successfully isolated from primary human ovarian tumors, which formed typical spheroids in serum-free medium and had stronger ability of tumorigenesis. The results of related experiments verified that CD133 positive cells owned the properties of cancer stem cells. The ovarian cancer stem cells presenting the characteristics of stemness in vitro and in vivo, have been successfully isolated from primary human ovarian tumor tissues by MACS. The isolated ovarian cancer stem cells could be used in future researches of their biological functions.

Immunohistochemical Expression of Stem Cell, Endothelial Cell, and Chemosensitivity Markers in Primary Glioma Spheroids Cultured in Serum-Containing and Serum-Free Medium

To investigate the influence of serum-free medium (SFM) supplemented with epidermal growth factor and basic fibroblast growth factor compared with conventional serum-containing medium (SCM) on the phenotype of organotypic primary spheroids from seven gliomas. Paraffin sections of the original surgical specimens, primary glioma spheroids, and U87 derived spheroids were stained immunohistochemically with the stem cell markers CD133, podoplanin, Sox2, Bmi-1, and nestin; the endothelial cell markers CD31, CD34, and Von Willebrand Factor (VWF); the chemosensitivity markers P-glycoprotein and tissue inhibitor of metalloproteinases-1 (TIMP-1); and glial fibrillary acidic protein, neural cell adhesion molecule CD56, and the proliferation marker Ki67. Scoring of the immunohistochemical stainings showed that the expression of CD133 and all other markers included was preserved in primary spheroids, confirming the in vivo-like nature of these spheroids. Spheroids in SFM better mimicked the in vivo phenotype with significantly more CD133, CD34, VWF, P-glycoprotein, TIMP-1, and Ki67 compared with SCM. In this first study of the influence of SFM on primary glioma spheroids, the conditions favored an in vivo-like phenotype with increased expression of CD133. More vascular structures were found in SFM, suggesting that the close relationship between blood vessels and tumor stem-like cells was better preserved in this medium.

Abstract 4249: Shape change induces nitric oxide and stemness in colorectal cancer cells

Abstract Shape changes in cancer cells profoundly alter the expression of genes that affect malignant properties. We postulated that shape change may increase expression of NANOG, an embryonic stem cell transcription factor expressed in human colorectal carcinoma (CRC) that regulates pluripotency and proliferation. Since shape change also increases intracellular nitric oxide (iNO) expression, we further postulated that iNO modulates NANOG expression. Clone A and CX-1 human CRC lines were cultured in serum-containing medium in monolayer cultures or in spheroids in serum free medium in low attachment plates. NANOG promoter activity was assessed in stable transfectants expressing promoter-driven GFP. iNO concentrations were determined by fluorescence cytometry with DAF-FM diacetate while Nanog gene and protein expression was analyzed by qRT-PCR and densitometry of western blots, respectively. Spherogenicity was measured by the percentage of spheroids formed from single cells in serum-free medium. Monolayer cultures with flat, spreading cells had low NANOG gene and protein expression, promoter activity and iNO whereas spheroids displayed high NANOG gene expression, promoter activity and iNO. 7 day monolayer cultures initiated with 104 cells/ml (Sparse) grew horizontally compared to cultures initiated with 106 cells/ml (Dense) that grew vertically and formed colonies. Sparse cultures had low NANOG gene expression, low NANOG promoter activity and low iNO. Dense cultures had high NANOG gene expression, NANOG promoter activity and iNO. Since NO stimulates cGMP production, the effects of the NO synthase inhibitor L-NMMA, NOC (a NO donor), 8-Br-cGMP (cGMP agonist) and ODQ (cGMP inhibitor) were tested on CRC monolayers. Physiologic concentrations of NOC increased NANOG and Oct4 gene and protein expression 3-fold in highly metastatic CX-1 cells but not in weakly metastatic Clone A cells. NOC also increased CD44 in CX-1 but not Clone A. cGMP may regulate NANOG expression since 8-Br-cGMP increased NANOG and Oct4 protein expression up to 3-fold in CX-1 but not in Clone A and without affecting CD44 expression. ODQ inhibited NANOG expression. L-NMMA stimulated both NANOG protein expression and the spherogenicity of CX-1 but not Clone A. NOC and 8-Br-cGMP increased inducible NO synthase (NOS2) but not NOS3 gene expression 3-fold in both spheroids and monolayers while ODQ decreased NOS2 levels. Assays are in progress to test the effects of NOC and the cGMP modulators on spherogenicity. Shape change, iNO and cGMP enhance NANOG, NOS2 and CD44 expression to alter CRC cancer stem cell function. cGMP may be an important regulator of stemness genes. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4249.

Cardiac neural crest cells contribute to the dormant multipotent stem cell in the mammalian heart.

Arodent cardiac side population cell fraction formed clonal spheroids in serum-free medium, which expressed nestin, Musashi-1, and multi-drug resistance transporter gene 1, markers of undifferentiated neural precursor cells. These markers were lost following differentiation, and were replaced by the expression of neuron-, glial-, smooth muscle cell–, or cardiomyocyte-specific proteins. Cardiosphere-derived cells transplanted into chick embryos migrated to the truncus arteriosus and cardiac outflow tract and contributed to dorsal root ganglia, spinal nerves, and aortic smooth muscle cells. Lineage studies using double transgenic mice encoding protein 0–Cre/Floxed-EGFP revealed undifferentiated and differentiated neural crest-derived cells in the fetal myocardium. Undifferentiated cells expressed GATA-binding protein 4 and nestin, but not actinin, whereas the differentiated cells were identified as cardiomyocytes. These results suggest that cardiac neural crest-derived cells migrate into the heart, remain there as dormant multipotent stem cells—and under the right conditions—differentiate into cardiomyocytes and typical neural crest-derived cells, including neurons, glia, and smooth muscle.

Three-dimensional culture of human tumor cells under standardized medium conditions.

The 3-dimensional culture of human tumor spheroids under standardized medium conditions may reveal information on specific biological parameters that could be masked in serum-supplemented media. Spheroids derived from human tumor cells are growth retarded in media free of serum. Ex-Cyte IV is a substance derived from human blood that can be used to improve growth in tissue culture. In this study the growth of spheroids from four different human tumor cell lines was studied when grown in medium free of serum, medium supplemented with varying concentrations Ex-Cyte IV, and medium supplemented with foetal calf serum (FCS). The parameters used for comparisons were growth rate, growth enhancement, clonogenicity and cell cycle distribution. The four cell lines showed different growth rates in serum-free medium, which were increased to different extents when Ex-Cyte IV or FCS were added. The growth enhancing effect induced by Ex-Cyte IV was differently concentration dependent for each cell line. The clonogenicity of cells grown as spheroids in serum-free medium was lower than in spheroids grown in supplemented media. There was no difference in clonogenicity between the differently supplemented media. All four cell lines responded to growth in serum-free medium with a drop in the S-phase and G2M phase. The present study provides a novel approach to the study of human tumor cells in 3-dimensional culture under defined conditions. The human serum derived substance Ex-Cyte IV may provide a method to obtain information on specific biological parameters that could be masked in serum-supplemented media.