Fibroblasts In Coculture System Research Articles

Drug-Resistance Biomarkers in Patient-Derived Colorectal Cancer Organoid and Fibroblast Co-Culture System.

Colorectal cancer, the third most commonly occurring tumor worldwide, poses challenges owing to its high mortality rate and persistent drug resistance in metastatic cases. We investigated the tumor microenvironment, emphasizing the role of cancer-associated fibroblasts in the progression and chemoresistance of colorectal cancer. We used an indirect co-culture system comprising colorectal cancer organoids and cancer-associated fibroblasts to simulate the tumor microenvironment. Immunofluorescence staining validated the characteristics of both organoids and fibroblasts, showing high expression of epithelial cell markers (EPCAM), colon cancer markers (CK20), proliferation markers (KI67), and fibroblast markers (VIM, SMA). Transcriptome profiling was conducted after treatment with anticancer drugs, such as 5-fluorouracil and oxaliplatin, to identify chemoresistance-related genes. Changes in gene expression in the co-cultured colorectal cancer organoids following anticancer drug treatment, compared to monocultured organoids, particularly in pathways related to interferon-alpha/beta signaling and major histocompatibility complex class II protein complex assembly, were identified. These two gene groups potentially mediate drug resistance associated with JAK/STAT signaling. The interaction between colorectal cancer organoids and fibroblasts crucially modulates the expression of genes related to drug resistance. These findings suggest that the interaction between colorectal cancer organoids and fibroblasts significantly influences gene expression related to drug resistance, highlighting potential biomarkers and therapeutic targets for overcoming chemoresistance. Enhanced understanding of the interactions between cancer cells and their microenvironment can lead to advancements in personalized medical research..

The anti-neoplastic effects of metformin modulate the acquired phenotype of fibroblast cells in the breast cancer-normal fibroblast co-culture system.

Intracellular communications between breast cancer and fibroblast cells were reported to be involved in cancer proliferation, growth, and therapy resistance. The hallmarks of cancer-fibroblast interactions, consisting of caveolin 1 (Cav1) and mono-carboxylate transporter 4 (MCT4) (metabolic coupling markers), along with IL-6, TGFβ, and lactate secretion, are considered robust biomarkers predicting recurrence and metastasis. In order to promote a novel phenotype in normal fibroblasts, we predicted that breast cancer cells could be able to cause loss of Cav1 and increase of MCT4, as well as elevate IL-6 and TGFβ in nearby normal fibroblasts. We created a co-culture model using breast cancer (4T1) and normal fibroblast (NIH3T3) cell lines cultured under specific experimental conditions in order to directly test our theory. Moreover, we show that long-term co-culture of breast cancer cells and normal fibroblasts promotes loss of Cav1 and gain of MCT4 in adjacent fibroblasts and increase lactate secretion. These results were validated using the monoculture of each group separately as a control. In this system, we show that metformin inhibits IL-6 and TGFβ secretion and re-expresses Cav1 in both cells. However, MCT4 and lactate stayed high after treatment with metformin. In conclusion, our work shows that co-culture with breast cancer cells may cause significant alterations in the phenotype and secretion of normal fibroblasts. Metformin, however, may change this state and affect fibroblasts' acquired phenotypes. Moreover, mitochondrial inhibition by metformin after 8 days of treatment, significantly hinders tumor growth in mouse model of breast cancer.

Establishment and Characterization of an HBV Viral Spread and Infectious System following Long-Term Passage of an HBV Clinical Isolate in the Primary Human Hepatocyte and Fibroblast Coculture System.

The existing cell culture-based methods to study hepatitis B virus (HBV) have limitations and do not allow for viral long-term passage. The aim of this study was to develop a robust in vitro long-term viral passage system with optimized cell culture conditions and a viral isolate with the ability to spread and passage. An HBV genotype A clinical isolate was subjected to multiple rounds of UV treatment and passaged in an optimized primary human hepatocyte (PHH)/human fibroblast coculture system. The passaged UV-treated virus was sequenced and further characterized. In addition, a panel of mutant viruses containing different combinations of mutations observed in this virus was investigated. The clinical isolate was passaged for 20 rounds with 21 days per round in an optimized PHH/human fibroblast coculture system while subject to UV mutagenesis. This passaged UV-mutated isolate harbored four mutations: G225A (sR24K) in the S gene, A2062T in the core gene, and two mutations G1764A and C1766T (xV131I) in the basal core promoter (BCP) region. In vitro characterization of the four mutations suggested that the two BCP mutations G1764A and C1766T contributed to the increased viral replication and viral infectivity. A robust in vitro long-term HBV viral passage system has been established by passaging a UV-treated clinical isolate in an optimized PHH/fibroblast coculture system. The two BCP mutations played a key role in the virus's ability to passage. This passage system can be used for studying the entire life cycle of HBV and has the potential for in vitro drug-resistance selection upon further optimization. IMPORTANCE The existing cell culture-based methods to study HBV have limitations and do not allow for viral long-term passage. In this study, an HBV genotype A clinical isolate was subjected to multiple rounds of UV treatment and passaged in an optimized PHH/human fibroblast coculture system. This passaged UV-mutated isolate carried four mutations across the HBV genome, and in vitro characterization of the four mutations suggested that the two basal core promoter (BCP) mutations G1764A and C1766T played a key role in the virus's ability to passage. In summary, we have developed a robust in vitro long-term HBV viral passage system by passaging an UV-treated HBV genotype A clinical isolate in an optimized PHH/human fibroblast coculture system. This passage system can be used for studying the entire life cycle of HBV and has the potential for in vitro drug-resistance selection upon further optimization.

Modeling macrophage response to periodontal infections in vitro

Periodontal disease (PD) is a chronic inflammatory disorder characterized by the destruction of connective tissue, tooth loss and systemic infections. Numerous virulence factors derived from chronic opportunistic pathogens such as Porphyromonas gingivalis are responsible for enhanced microbial colonization, stimulation of inflammatory responses, and suppression of host defense response. Individuals with PD present with a complex array of inflammatory mediators, and cellular infiltration by neutrophils, lymphocytes and monocytes/macrophages. Although macrophages comprise 5–30% of cells in the cellular infiltrate of human periodontal lesions, how macrophage skewing contributes to PD, and more specifically in the downstream development of a macrophage immune response to oral bacteria are unclear. We, including others have investigated the activation of monocytes/macrophages, and discovered the means of type I vs. type II inflammatory activation vs. deactivation of macrophages both in vitro and in vivo. We have shown previously that P. gingivalis infection induces M1 macrophage formation, whereas opsonized‐P. gingivalis challenge was able to switch to M2‐like phenotype demonstrating macrophage plasticity. However, the mechanism of modulation and kinetics of macrophage inflammatory reactions during the process is unclear. Prior research in the field has failed to appreciate that it is the same monocyte/macrophage cell population which is initially polarized towards an effector “inflammatory” program and subsequently re‐polarized to the deactivation program not just in PD but in any inflammation. The overarching goal of our research is to understand how macrophage skewing contributes to PD, and the downstream development of immune response to PD‐associated bacteria towards development of new macrophage‐based therapeutics. Since macrophage polarization dynamics cannot be studied in an animal model, a reliable in vitro system is needed to provide fundamental insights into macrophage biology. We have developed an in vitro model that recapitulates different phases of the inflammatory response (recruitment, initiation, development and resolution), based on purified human peripheral blood monocytes exposed to a battery of microenvironmental cues mimicking macrophage polarization/depolarization in vivo and we have created a kinetic profile of polarization by RNA analysis. Further we have applied our model to macrophage/gingival fibroblast coculture system to study kinetics of polarization. And by comparing gene clusters that are differentially expressed in M1 and M2, we have successfully recapitulated the macrophage polarization kinetics of acute inflammatory and resolution phases during periodontal inflammation and validated the key inflammatory and transcriptional factors using RT‐PCR at different phases. In conclusion, we developed an in vitro model which can be further employed to test novel macrophage‐based therapeutics to treat periodontal disease.

M2 macrophage-mediated interleukin-4 signalling induces myofibroblast phenotype during the progression of benign prostatic hyperplasia

Benign prostatic hyperplasia (BPH) is a progressive disease in elderly men, but potential factors accelerating its progression remain largely unknown. The aim of this study was to elucidate the factors affecting BPH progression by understanding the complex mechanisms causing early- progressed BPH, which progresses rapidly and requires surgical intervention before the age of 50. Three groups of human prostate tissue samples, from patients with early-progressed BPH, age-matched prostate and elderly BPH tissues, were collected (n = 25 each). We compared these tissues to determine the histologic features and molecular mechanisms underlying BPH progression. We found that early-progressed BPH samples were characterised by aberrant stromal hyper-proliferation, collagen deposition and increased M2 macrophage infiltration, compared to those from age-matched prostate and elderly BPH tissues. The M2 macrophage–fibroblast co-culture system demonstrated that the myofibroblast phenotypes were strongly induced only in fibroblasts from the early-progressed BPH samples, while the co-cultured M2 macrophages expressed high levels of pro-fibrotic cytokines, such as IL4 and TGFβ1. M2 macrophage-derived IL4, but not TGFβ1, selectively induced the myofibroblast phenotype through the JAK/STAT6, PI3K/AKT and MAPK/ERK signalling pathways in the early-progressed BPH prostate fibroblasts. Taken together, our results indicate that induction of the myofibroblast phenotype may lead to BPH progression through M2 macrophage-mediated IL4 signalling, and that IL4 may represent a potential therapeutic target, allowing the prevention of M2 macrophage activation and fibroblast-to-myofibroblast differentiation.

Genome-wide DNA methylation analysis in lung fibroblasts co-cultured with silica-exposed alveolar macrophages

BackgroundExposure to crystalline silica is considered to increase the risk of lung fibrosis. The primary effector cell, the myofibroblast, plays an important role in the deposition of extracellular matrix (ECM). DNA methylation change is considered to have a potential effect on myofibroblast differentiation. Therefore, the present study was designed to investigate the genome-wide DNA methylation profiles of lung fibroblasts co-cultured with alveolar macrophages exposed to crystalline silica in vitro.MethodsAM/fibroblast co-culture system was established. CCK8 was used to assess the toxicity of AMs. mRNA and protein expression of collagen I, α-SMA, MAPK9 and TGF-β1 of fibroblasts after AMs exposed to 100 μg /ml SiO2 for 0–, 24–, or 48 h were determined by means of quantitative real-time PCR, immunoblotting and immunohistochemistry. Genomic DNA of fibroblasts was isolated using MeDIP-Seq to sequence. R software, GO, KEGG and Cytoscape were used to analyze the data.ResultsSiO2 exposure increased the expression of collagen I and α-SMA in fibroblasts in co-culture system. Analysis of fibroblast methylome identified extensive methylation changes involved in several signaling pathways, such as the MAPK signaling pathway and metabolic pathways. Several candidates, including Tgfb1 and Mapk9, are hubs who can connect the gene clusters. MAPK9 mRNA expression was significantly higher in fibroblast exposed to SiO2 in co-culture system for 48 h. MAPK9 protein expression was increased at both 24-h and 48-h treatment groups. TGF-β1 mRNA expression of fibroblast has a time-dependent manner, but we didn’t observe the TGF-β1 protein expression.ConclusionTgfb1 and Mapk9 are helpful to explore the mechanism of myofibroblast differentiation. The genome-wide DNA methylation profiles of fibroblasts in this experimental silicosis model will be useful for future studies on epigenetic gene regulation during myofibroblast differentiation.

ADAM‐10 is overexpressed in rheumatoid arthritis synovial tissue and mediates angiogenesis

To examine the expression of ADAM-10 in rheumatoid arthritis (RA) synovial tissue (ST) and the role it plays in angiogenesis. ADAM-10 expression was determined using immunohistology, Western blotting, and quantitative polymerase chain reaction. In order to examine the role of ADAM-10 in angiogenesis, we performed in vitro Matrigel tube formation and chemotaxis assays using human microvascular endothelial cells (HMVECs) transfected with control or ADAM-10 small interfering RNA (siRNA). To determine whether ADAM-10 plays a role in angiogenesis in the context of RA, we performed Matrigel assays using a coculture system of HMVECs and RA synovial fibroblasts. Endothelial cells and lining cells within RA ST expressed high levels of ADAM-10 compared with cells within osteoarthritis ST and normal ST. ADAM-10 expression was significantly elevated at the protein and messenger RNA levels in HMVECs and RA synovial fibroblasts stimulated with proinflammatory mediators compared with unstimulated cells. ADAM-10 siRNA-treated HMVECs had decreased endothelial cell tube formation and migration compared with control siRNA-treated HMVECs. In addition, ADAM-10 siRNA-treated HMVECs from the RA synovial fibroblast coculture system had decreased endothelial cell tube formation compared with control siRNA-treated HMVECs. These data show that ADAM-10 is overexpressed in RA and suggest that ADAM-10 may play a role in RA angiogenesis. ADAM-10 may be a potential therapeutic target in inflammatory angiogenic diseases such as RA.

Hepatocyte growth factor and other fibroblast secretions modulate the phenotype of human bronchial epithelial cells

The luminal airway surface is lined with epithelial cells that provide a protective barrier from the external environment and clear inhaled pathogens from the lung. To accomplish this important function, human bronchial epithelial (HBE) cells must be able to rapidly regenerate a mucociliary layer of cells following epithelial injury. Whereas epithelial-fibroblast interactions are known to modulate the airway architecture during lung development and repair, little is known about how these two cells interact. Using a primary HBE and lung fibroblast coculture system, we demonstrate that 1) subepithelial fibroblasts provide a suitable environment for differentiation of HBE cells into a polarized ciliated phenotype despite being cultured in media that induces terminal squamous differentiation and growth arrest in the absence of fibroblasts, 2) HBE cells cocultured with subepithelial fibroblasts exhibit augmented ciliogenesis, accelerated wound repair, and diminished polarized ion transport compared with cells grown in control conditions, and 3) hepatocyte growth factor (HGF) is important for subepithelial fibroblast modulation of HBE cell differentiation. These results provide a model to study fibroblast modulation of epithelial phenotype and indicate that HGF secreted by subepithelial fibroblasts contributes to HBE cell differentiation.

The effect of tubular epithelial cells activated by aldosterone on renal interstitial fibroblasts in co-culture system

Local aldosterone (ALDO) could be synthesized by human proximal tubular epithelial cell lines (HKC) after the stimulation of endothelin-1 (ET-1) in vitro. T to observe the effect of tubular epithelial cells activated by aldosterone (ALDO) on renal interstitial fibroblasts in co-culture system. (1) Human Proximal tubular epithelial cells of the line HKC were stimulated with ALDO at different concentrations and times, then reverse transcription polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA) were performed to detect TGF-beta1 expression; (2) HKC were co-stimulated with ET-1 (10(-9) mol/L) and spironolactone at different concentrations and times to evaluate the influence of endogenous ALDO on TGF-beta1 expression; (3) HKC which were activated by 10(-7) mol/L ALDO for 12 h, and hRIFs were co-cultured for 48 h with or without anti-TGF-beta1 antibody (1.0, 2.0 microg/ml) in the media, then the production of type I collagen (Col-I) in the cell layer of hRIFs was detected by ELISA. (1) After stimulation with ALDO, the expression of TGF-beta1 by HKC was up-regulated in a dose-and time-dependent manner. With 10(-9) or 10(-7) mol/L ALDO stimulation (mRNA determination at 12 h and protein at 48 h), the expression of TGF-beta1 was significantly increased (vs 0 mol/L ALDO, P < 0.05 or 0.01). With 10(-7) mol/L ALDO stimulated at different times (mRNA determination at 8 h, 12 h and 16 h and protein at 12 h, 24 h and 48 h), the expression of TGF-beta1 was also significantly increased (vs 0 h, P < 0.05 or 0.01). (2) After co-stimulation with ET-1 and spironolatone, the expression of TGF-beta1 by HKC was down-regulated in a dose-and-time dependent manner along with spironolatone. The expression of TGF-beta1 mRNA and protein was decreased in the 10(-9) or 10(-7) mol/L spironolatone groups compared with 0 mol/L group (P < 0.05 or 0.01). The expression of TGF-beta1 (mRNA and protein) was significantly decreased in 10(-9) mol/L ET-1 and 10(-7) mol/L spironolatone co-stimulation group compared with 10(-9) mol/L ET-1 stimulated group. (3) The production of Col-I by hRIFs which were co-cultured with activated HKC by ALDO, was significantly increased (vs control group in which hRIFs co-cultured with normal HKC, P < 0.01), and this effect was partially inhibited by 1.0 or 2.0 microg/ml anti-TGF-beta1 antibody (P < 0.05). The expression of TGF-beta1 in HKC cells can not only be up-regulated by exogenous ALDO, but also can be up-regulated by endogenous ALDO in an autocrine manner. The HKC cells activated by ALDO can promote the synthesis of Col-I in hRIFs by a "cross talking" way, and this effect is partially mediated by TGF-beta1.

Long-term function of cryopreserved rat hepatocytes in a coculture system.

The goal of this study was to investigate postpreservation long-term function of cryopreserved primary rat hepatocytes using the hepatocyte/3T3-J2 fibroblast coculture system. The long-term function of thawed hepatocytes cocultured with fibroblasts was evaluated and compared with hepatocytes cultured without fibroblasts. Fresh isolated primary rat hepatocytes were frozen at a controlled rate (-1 degrees C/min) up to -80 degrees C, and then stored in liquid nitrogen for up to 90 days. Thawed hepatocytes were thereafter cocultured with 3T3-J2 murine fibroblasts and cocultivation was monitored for 14 days. The viability of fresh isolated hepatocytes was 91.4%, and that of cryopreserved hepatocytes was 82.1%. Cellular morphology and polarity, which were determined by the localization of actin filaments and connexin-32, were successfully maintained in cryopreserved hepatocytes following cryopreservation. Albumin and urea synthesis reached the maximum level and became stable after day 7 in coculture in both fresh and cryopreserved hepatocytes. Urea synthesis of cryopreserved hepatocytes was maintained 89.0% of nonfrozen fresh control, and albumin production of cryopreserved hepatocytes was 63.7% of control in coculture. Cytochrome P450 activity, which was measured by deethylation of ethoxyresorufin, was also maintained in cryopreserved hepatocytes at 88.6% of nonfrozen fresh control in coculture. The retention of synthetic and detoxification activities was verified to be well preserved during extended low-temperature storage (90 days). Both fresh control and cryopreserved hepatocytes cultured without fibroblast did not retain their synthetic and detoxification functions in long-term culture. These data illustrate that, through the utilization of our cryopreservation procedure, primary hepatocyte function was successfully maintained when placed into coculture configuration following thawing.

Induction of tube formation by angiopoietin-1 in endothelial cell/fibroblast co-culture is dependent on endogenous VEGF.

The angiopoietin-1 (Ang1)/Tie2 receptor system is known to be important for angiogenesis and vascular remodeling. However, its contribution to the survival and morphogenesis of endothelial cells is still not well elucidated. In this study, we analyzed the role of the Ang1/Tie2 pathway in cell survival and tube formation using a human umbilical vein endothelial (HUVE) cell and fibroblast co-culture system. In this system, which mimics angiogenesis in vivo, fibroblasts secrete a basal level of vascular endothelial growth factor (VEGF), and Ang1 stimulated tube formation. However, anti-VEGF or anti-VEGF receptor-2 neutralizing antibody blocked the Ang1-induced tube formation. Furthermore, other angiogenic factors such as hepatic growth factor (HGF) and basic fibroblast growth factor (bFGF) showed the same phenotype as Ang1, i.e., a stimulatory effect only in the presence of endogenous VEGF. The Ang1-promoted tube formation was mainly due to suppression of HUVE cell apoptosis in a PI3-kinase-dependent manner. These findings suggest that Ang1 stimulates tube formation in vivo via the PI3-kinase/Akt pathway, but this effect takes place only in a VEGF-dependent manner.

The IL-6 family cytokines, interleukin-6, interleukin-11, oncostatin M, and leukemia inhibitory factor, enhance mast cell growth through fibroblast-dependent pathway in mice.

Mast cell hyperplasia is observed in various inflammatory skin diseases. Although the mechanisms involved in the pathogenesis of these conditions remains largely uninvestigated, it is speculated that mediators produced in the lesional skin provide a favorable microenvironment for mast cell growth. Among the proinflammatory mediators, leukemia inhibitory factor (LIF), which shares a receptor component (gp130 subunit) with interleukin-6 (IL-6), has been identified as a mast cell growth-enhancing factor produced by cells of the keratinocyte-derived cell line (KCMH-1). In this study, we investigated the effect of four IL-6 family cytokines, IL-6, IL-11, oncostatin M (OSM) and LIF on mast cell growth in a mast cell/fibroblast co-culture system. When mouse bone marrow-derived cultured mast cells (BMMC) were maintained on a NIH/3T3 fibroblast monolayer, these cytokines induced proliferation of the mast cells, but none of the cytokines had any effect on mast cell proliferation in the absence of fibroblasts. mRNA for gp130 and receptors for the four IL-6 family cytokines were detected in NIH/3T3 fibroblasts by reverse transcriptase-mediated polymerase chain reaction. In contrast, only mRNA for the IL-11 receptor and gp130 were detected in BMMC. Tyrosine phosphorylation of gp130 was observed in NIH/3T3 fibroblasts after stimulation with all the cytokines. Some IL-6 family cytokines enhanced the production of stem cell factor (SCF), a potent mast cell growth factor, from NIH/3T3 fibroblasts, but the amount of SCF produced by NIH/3T3 fibroblasts was not paralleled by the mast cell growth-enhancement induced by the IL-6 family cytokines. When anti-SCF antibody was added with the IL-6 family cytokines in the BMMC/fibroblast coculture system, a significant effect of these cytokines remained, although the growth-enhancing activity was markedly reduced. A similar result was obtained when BMMC were prepared from W/W(V)-mice, which lack functional c-kit, in the BMMC/ fibroblast coculture system. These results suggest that IL-6 family cytokines stimulate mast cell growth by a fibroblast-dependent mechanism, and also suggest the existence of another pathway between BMMC and NIH/3T3 fibroblasts cooperating with the SCF/c-kit pathway. IL-6 family cytokines may thus contribute to mast cell hyperplasia in skin diseases.

Leukemia inhibitory factor enhances mast cell growth in a mast cell/fibroblast co-culture system through Stat3 signaling pathway of fibroblasts

Leukemia inhibitory factor (LIF) enhanced mast cell growth in a mast cell/3T3 fibroblast co-culture system, however the precise mechanisms have not been defined. Western blot analysis showed that bone marrow-derived mast cells failed to express both LIF receptor (LIFR) and gp130, whereas 3T3 fibroblasts expressed both LIFR and gp130. This result indicates that the activity of LIF for mast cell growth is mediated by 3T3 fibroblasts. Signal transducer and activator of transcription (Stat) 3-transfected 3T3 fibroblasts enhanced mast cell growth. In addition, dominant-negative Stat3-transfected fibroblasts blocked LIF-mediated mast cell growth in the co-culture system. In conclusion, LIF-induced mast cell growth in the co-culture system is mediated by an indirect pathway via 3T3 fibroblasts through activating Stat3 signaling pathway in 3T3 fibroblasts.

Nitric oxide released from Swiss 3T3 fibroblasts acts as a cytostatic agent for cultured mast cells.

Nitric oxide (NO) released from Swiss 3T3 fibroblasts inhibited the proliferation of cultured mast cells (CMCs) on the CMCs/Swiss 3T3 fibroblast co-culture system. Swiss 3T3 fibroblasts produced NO upon treatment with recombinant interferon-gamma (rIFN-gamma). The production of NO was markedly increased by the co-treatment of rIFN-gamma and recombinant tumor necrosis factor-alpha. This production was dependent on L-arginine and could be inhibited by the competitive substrate inhibitor, L-arginine analogue NG-monomethyl-L-arginine (NGMMA). In addition, the number of CMCs increased when treated with NGMMA in the co-culture system. These findings indicate that the elevated production of NO from Swiss 3T3 fibroblasts during the co-culture inhibited the proliferation of CMCs.

Candidate ligand for the c-kit transmembrane kinase receptor: KL, a fibroblast derived growth factor stimulates mast cells and erythroid progenitors.

The c-kit proto-oncogene encodes a transmembrane tyrosine kinase receptor for an unidentified ligand and is allelic with the murine white-spotting locus (W). W mutations affect melanogenesis, gametogenesis and hematopoiesis during development and in adult life. Cellular targets of W mutations in hematopoiesis include distinct cell populations in the erythroid and mast cell lineages as well as stem cells. In the absence of interleukin-3 (IL-3) mast cells derived from normal mice but not from W mutant mice can be maintained by co-culture with 3T3 fibroblasts. Based on the defective proliferative response of W mast cells in the 3T3 fibroblast co-culture system it had been proposed that fibroblasts produce the c-kit ligand. We have used a mast cell proliferation assay to purify a 30 kd protein, designated KL, from conditioned medium of Balb/3T3 fibroblasts to apparent homogeneity. KL stimulates the proliferation of normal bone marrow derived mast cells but not mast cells from W mice, although both normal and mutant mast cells respond similarly to IL-3. Connective tissue-type mast cells derived from the peritoneal cavity of normal mice were found to express a high level of c-kit protein on their surface and to proliferate in response to KL. The effect of KL on erythroid progenitor cells was investigated as well. In combination with erythropoietin, KL was found to stimulate early erythroid progenitors (BFU-E) from fetal liver and spleen cells but not from bone marrow cells of adult mice and from fetal liver cells of W/W mice.(ABSTRACT TRUNCATED AT 250 WORDS)