RESUMO
Reliable in vitro to in vivo translation of cytochrome P450 (CYP) 3A4 induction potential is essential to support risk mitigation for compounds during pharmaceutical discovery and development. In this study, a linear correlation of CYP3A4 mRNA induction potential in human hepatocytes with the respective pregnane-X receptor (PXR) activation in a reporter gene assay using DPX2 cells was successfully demonstrated for 13 clinically used drugs. Based on this correlation, using rifampicin as a positive control, the magnitude of CYP3A4 mRNA induction for 71 internal compounds at several concentrations up to 10 µM (n = 90) was predicted within 2-fold error for 64% of cases with only a few false positives (19%). Furthermore, the in vivo area under the curve reduction of probe CYP substrates was reasonably predicted for eight marketed drugs (carbamazepine, dexamethasone, enzalutamide, nevirapine, phenobarbital, phenytoin, rifampicin, and rufinamide) using the static net effect model using both the PXR activation and CYP3A4 mRNA induction data. The liver exit concentrations were used for the model in place of the inlet concentrations to avoid false positive predictions and the concentration achieving twofold induction (F2) was used to compensate for the lack of full induction kinetics due to cytotoxicity and solubility limitations in vitro. These findings can complement the currently available induction risk mitigation strategy and potentially influence the drug interaction modeling work conducted at clinical stages. SIGNIFICANCE STATEMENT: The established correlation of CYP3A4 mRNA in human hepatocytes to PXR activation provides a clear cut-off to identify a compound showing an in vitro induction risk, complementing current regulatory guidance. Also, the demonstrated in vitro-in vivo translation of induction data strongly supports a clinical development program although limitations remain for drug candidates showing complex disposition pathways, such as involvement of auto-inhibition/induction, active transport and high protein binding.
Assuntos
Citocromo P-450 CYP3A , Receptores de Esteroides , Humanos , Citocromo P-450 CYP3A/metabolismo , Receptor de Pregnano X/metabolismo , Receptores de Esteroides/genética , Receptores de Esteroides/metabolismo , Sistema Enzimático do Citocromo P-450/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Rifampina/farmacologia , Rifampina/metabolismo , Indução Enzimática , Hepatócitos/metabolismo , RNA Mensageiro/metabolismoRESUMO
The presence of microbes in the colon impacts host physiology. Therefore, microbes are being evaluated as potential treatments for colorectal diseases. Humanized model systems that enable robust culture of primary human intestinal cells with bacteria facilitate evaluation of potential treatments. Here, we describe a protocol that can be used to coculture a primary human colon monolayer with aerotolerant bacteria. Primary human colon cells maintained as organoids are dispersed into single-cell suspensions and then seeded on collagen-coated Transwell inserts, where they attach and proliferate to form confluent monolayers within days of seeding. The confluent monolayers are differentiated for an additional 4 d and then cocultured with bacteria. As an example application, we describe how to coculture differentiated colon cells for 8 h with four strains of Bacteroides thetaiotaomicron, each engineered to detect different colonic microenvironments via genetically embedded logic circuits incorporating deoxycholic acid and anhydrotetracycline sensors. Characterization of this coculture system reveals that barrier function remains intact in the presence of engineered B. thetaiotaomicron. The bacteria stay close to the mucus layer and respond in a microenvironment-specific manner to the inducers (deoxycholic acid and anhydrotetracycline) of the genetic circuits. This protocol thus provides a useful mucosal barrier system to assess the effects of bacterial cells that respond to the colonic microenvironment, and may also be useful in other contexts to model human intestinal barrier properties and microbiota-host interactions.
Assuntos
Bacteroides thetaiotaomicron/fisiologia , Colo/citologia , Células Epiteliais/fisiologia , Mucosa Intestinal/citologia , Técnicas de Cocultura/métodos , Humanos , OrganoidesRESUMO
Intestinal homeostasis is tightly regulated by the orchestrated actions of a multitude of cell types, including enterocytes, goblet cells, and immune cells. Disruption of intestinal barrier function can increase susceptibility to pathogen invasion and destabilize commensal microbial-epithelial-immune interaction, manifesting in various intestinal and systemic pathologies. However, a quantitative understanding of how these cell types communicate and collectively contribute to tissue function in health and disease is lacking. Here, we utilized a human intestinal epithelial-dendritic cell model and multivariate analysis of secreted factors to investigate the cellular crosstalk in response to physiological and/or pathological cues (e.g., endotoxin, nonsteroidal anti-inflammation drug (NSAID)). Specifically, we demonstrated that treatment with diclofenac (DCF), an NSAID commonly used to treat inflammation associated with acute infection and other conditions, globally suppressed cytokine secretion when dosed in isolation. However, the disruption of barrier function induced by DCF allowed for luminal lipopolysaccharide (LPS) translocation and activation of resident immune cells that overrode the anti-inflammatory influence of DCF. DCF-facilitated inflammation in the presence of LPS was in part mediated by upregulation of macrophage migration inhibitory factor (MIF), an important regulator of innate immunity. However, while neutralization of MIF activity normalized inflammation, it did not lead to intestinal healing. Our data suggest that systems-wide suppression of inflammation alone is insufficient to achieve mucosal healing, especially in the presence of DCF, the target of which, the COX-prostaglandin pathway, is central to mucosal homeostasis. Indeed, DCF removal postinjury enabled partial recovery of intestinal epithelium functions, and this recovery phase was associated with upregulation of a subset of cytokines and chemokines, implicating their potential contribution to intestinal healing. The results highlight the utility of an intestinal model capturing immune function, coupled with multivariate analysis, in understanding molecular mechanisms governing response to microbial factors, supporting application in studying host-pathogen interactions.
Assuntos
Diclofenaco , Endotoxinas , Células Epiteliais , Humanos , Inflamação , Mucosa IntestinalRESUMO
After two decades teetering at the intersection of laboratory tool and therapeutic reality, with two siRNA drugs now clinically approved, this modality has finally come into fruition. Consistent with other emerging modalities, initial proof-of-concept efforts concentrated on coupling pharmacologic efficacy with desirable safety profiles. Consequently, thorough investigations of siRNA absorption, distribution, metabolism, and excretion (ADME) properties are lacking. Advancing ADME knowledge will aid establishment of in vitro-in vivo correlations and pharmacokinetic-pharmacodynamic relationships to optimize candidate selection through discovery and translation. Here, we outline the emerging siRNA design principles and discuss the consequences for siRNA disposition and biotransformation. We propose a conceptual framework for siRNA ADME evaluation, contextualizing the site of biotransformation product formation with PK-PD modulation, and end with a discussion around safety and regulatory considerations and future directions for this modality.
Assuntos
Biotransformação , Desenho de Fármacos , Desenvolvimento de Medicamentos , Avaliação Pré-Clínica de Medicamentos , Preparações Farmacêuticas/química , RNA Interferente Pequeno/química , Animais , Humanos , Preparações Farmacêuticas/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/farmacocinética , Distribuição TecidualRESUMO
Over the last decade, progress has been made on the development of microphysiological systems (MPS) for absorption, distribution, metabolism, and excretion (ADME) applications. Central to this progress has been proof of concept data generated by academic and industrial institutions followed by broader characterization studies, which provide evidence for scalability and applicability to drug discovery and development. In this review, we describe some of the advances made for specific tissue MPS and outline the desired functionality for such systems, which are likely to make them applicable for practical use in the pharmaceutical industry. Single organ MPS platforms will be valuable for modelling tissue-specific functions. However, dynamic organ crosstalk, especially in the context of disease or toxicity, can only be obtained with the use of inter-linked MPS models which will enable scientists to address questions at the intersection of pharmacokinetics (PK) and efficacy, or PK and toxicity. In the future, successful application of MPS platforms that closely mimic human physiology may ultimately reduce the need for animal models to predict ADME outcomes and decrease the overall risk and cost associated with drug development.
Assuntos
Dispositivos Lab-On-A-Chip , Técnicas Analíticas Microfluídicas , Preparações Farmacêuticas/metabolismo , Animais , Desenvolvimento de Medicamentos , Avaliação Pré-Clínica de Medicamentos , Indústria Farmacêutica , Humanos , Técnicas Analíticas Microfluídicas/instrumentação , Preparações Farmacêuticas/químicaRESUMO
An in vitro gut-immune co-culture model with apical and basal accessibility, designed to more closely resemble a human intestinal microenvironment, was employed to study the role of the N-linked protein glycosylation pathway in Campylobacter jejuni pathogenicity. The gut-immune co-culture (GIC) was developed to model important aspects of the human small intestine by the inclusion of mucin-producing goblet cells, human enterocytes and dendritic cells, bringing together a mucus-containing epithelial monolayer with elements of the innate immune system. The utility of the system was demonstrated by characterizing host-pathogen interactions facilitated by N-linked glycosylation, such as host epithelial barrier functions, bacterial invasion and immunogenicity. Changes in human intestinal barrier functions in the presence of 11168 C. jejuni (wildtype) strains were quantified using GICs. The glycosylation-impaired strain 11168 ΔpglE was 100-fold less capable of adhering to and invading this intestinal model in cell infectivity assays. Quantification of inflammatory signaling revealed that 11168ΔpglE differentially modulated inflammatory responses in different intestinal microenvironments, suppressive in some but activating in others. Virulence-associated outer membrane vesicles produced by wildtype and 11168ΔpglE C. jejuni were shown to have differential composition and function, with both leading to immune system activation when provided to the gut-immune co-culture model. This analysis of aspects of C. jejuni infectivity in the presence and absence of its N-linked glycome is enabled by application of the gut-immune model, and we anticipate that this system will be applicable to further studies of C. jejuni and other enteropathogens of interest.
Assuntos
Campylobacter jejuni/imunologia , Técnicas de Cocultura , Microbioma Gastrointestinal/imunologia , Interações Hospedeiro-Patógeno/imunologia , Polissacarídeos/imunologia , Animais , Humanos , Polissacarídeos/químicaRESUMO
Microphysiological systems (MPSs) are in vitro models that capture facets of in vivo organ function through use of specialized culture microenvironments, including 3D matrices and microperfusion. Here, we report an approach to co-culture multiple different MPSs linked together physiologically on re-useable, open-system microfluidic platforms that are compatible with the quantitative study of a range of compounds, including lipophilic drugs. We describe three different platform designs - "4-way", "7-way", and "10-way" - each accommodating a mixing chamber and up to 4, 7, or 10 MPSs. Platforms accommodate multiple different MPS flow configurations, each with internal re-circulation to enhance molecular exchange, and feature on-board pneumatically-driven pumps with independently programmable flow rates to provide precise control over both intra- and inter-MPS flow partitioning and drug distribution. We first developed a 4-MPS system, showing accurate prediction of secreted liver protein distribution and 2-week maintenance of phenotypic markers. We then developed 7-MPS and 10-MPS platforms, demonstrating reliable, robust operation and maintenance of MPS phenotypic function for 3 weeks (7-way) and 4 weeks (10-way) of continuous interaction, as well as PK analysis of diclofenac metabolism. This study illustrates several generalizable design and operational principles for implementing multi-MPS "physiome-on-a-chip" approaches in drug discovery.
Assuntos
Técnicas de Cocultura/métodos , Diclofenaco/farmacocinética , Dispositivos Lab-On-A-Chip , Fígado/metabolismo , Animais , Avaliação Pré-Clínica de Medicamentos , Humanos , Procedimentos Analíticos em Microchip , Modelos Biológicos , Fenótipo , RatosRESUMO
Investigation of the pharmacokinetics (PK) of a compound is of significant importance during the early stages of drug development, and therefore several in vitro systems are routinely employed for this purpose. However, the need for more physiologically realistic in vitro models has recently fueled the emerging field of tissue-engineered 3D cultures, also referred to as organs-on-chips, or microphysiological systems (MPSs). We have developed a novel fluidic platform that interconnects multiple MPSs, allowing PK studies in multi-organ in vitro systems along with the collection of high-content quantitative data. This platform was employed here to integrate a gut and a liver MPS together in continuous communication, and investigate simultaneously different PK processes taking place after oral drug administration in humans (e.g., intestinal permeability, hepatic metabolism). Measurement of tissue-specific phenotypic metrics indicated that gut and liver MPSs can be fluidically coupled with circulating common medium without compromising their functionality. The PK of diclofenac and hydrocortisone was investigated under different experimental perturbations, and results illustrate the robustness of this integrated system for quantitative PK studies. Mechanistic model-based analysis of the obtained data allowed the derivation of the intrinsic parameters (e.g., permeability, metabolic clearance) associated with the PK processes taking place in each MPS. Although these processes were not substantially affected by the gut-liver interaction, our results indicate that inter-MPS communication can have a modulating effect (hepatic metabolism upregulation). We envision that our integrative approach, which combines multi-cellular tissue models, multi-MPS platforms, and quantitative mechanistic modeling, will have broad applicability in pre-clinical drug development.
Assuntos
Diclofenaco/farmacocinética , Hidrocortisona/farmacocinética , Mucosa Intestinal/metabolismo , Fígado/metabolismo , Humanos , Técnicas In VitroRESUMO
Tissue-engineered heart valves are promising alternatives to address the limitations of current valve replacements, particularly for growing children. Current heart valve tissue engineering strategies involve the selection of biomaterial scaffolds, cell types, and often in vitro culture conditions aimed at regenerating a valve for implantation and subsequent maturation in vivo. However, identifying optimal combinations of cell sources, biomaterials, and/or bioreactor conditions to produce functional, durable valve tissue remains a challenge. Despite some short-term success in animal models, attempts to recapitulate aspects of the native heart valve environment based on 'best guesses' of a limited number of regulatory factors have not proven effective. Better outcomes for valve tissue regeneration will likely require a systems-level understanding of the relationships between multiple interacting regulatory factors and their effects on cell function and tissue formation. Until recently, conventional culture methods have not allowed for multiple design parameters to be considered at once. Emerging microtechnologies are well suited to systematically probe multiple inputs, in combination, in high throughput and with great precision. When combined with statistical and network systems analyses, these microtechnologies have excellent potential to define multivariate signal-response relationships and reveal key regulatory pathways for robust functional tissue regeneration.
Assuntos
Bioprótese , Doenças das Valvas Cardíacas , Próteses Valvulares Cardíacas , Regeneração , Engenharia Tecidual/métodos , Animais , Modelos Animais de Doenças , Doenças das Valvas Cardíacas/metabolismo , Doenças das Valvas Cardíacas/terapia , HumanosRESUMO
Previous studies demonstrated the importance of substrate stiffness and topography on the phenotype of many different cell types including fibroblasts. Yet the interaction of these two physical parameters remains insufficiently characterized, in particular for cardiac fibroblasts. Most studies focusing on contact guidance use rigid patterned substrates. It is not known how the ability of cardiac fibroblasts to follow grooves and ridges changes as the substrate stiffness is decreased to match the range of stiffness found in native heart tissues. This report demonstrates a significant interactive effect of substrate stiffness and topography on cardiac fibroblast elongation and orientation using polyacrylamide substrates of different stiffness and topography.
Assuntos
Resinas Acrílicas/síntese química , Materiais Biocompatíveis/síntese química , Colágeno/química , Fibroblastos/citologia , Resinas Acrílicas/farmacologia , Animais , Animais Recém-Nascidos , Materiais Biocompatíveis/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Elasticidade , Fibroblastos/efeitos dos fármacos , Fibroblastos/fisiologia , Compostos Heterocíclicos de 4 ou mais Anéis/farmacologia , Hidrogéis , Microscopia Eletrônica de Varredura , Miocárdio/citologia , Ratos , Propriedades de Superfície , Engenharia Tecidual , Alicerces TeciduaisRESUMO
Diseased tissues are noted for their compromised mechanical properties, which contribute to organ failure; regeneration entails restoration of tissue structure and thereby functions. Thus, the physical signature of a tissue is closely associated with its biological function. In this review, we consider a mechanics-centric view of disease and regeneration by drawing parallels between in vivo tissue-level observations and corroborative cellular evidence in vitro to demonstrate the importance of the mechanical stiffness of the extracellular matrix in these processes. This is not intended to devalue the importance of biochemical signaling; in fact, as we discuss, many mechanical stiffness-driven processes not only require cooperation with biochemical cues, but they ultimately converge at common signaling cascades to influence cell and tissue function in an integrative manner. The study of how physical and biochemical signals collectively modulate cell function not only brings forth a more holistic understanding of cell (patho)biology, but it also creates opportunities to control material properties to improve culture platforms for research and drug screening and aid in the rationale design of biomaterials for molecular therapy and tissue engineering applications.
Assuntos
Sistemas de Liberação de Medicamentos , Medicina Regenerativa/métodos , Engenharia Tecidual/métodos , Animais , Materiais Biocompatíveis/química , Técnicas de Cultura de Células , Elasticidade , Matriz Extracelular/metabolismo , Ensaios de Triagem em Larga Escala/métodos , Humanos , Células-Tronco/metabolismoRESUMO
OBJECTIVE: In calcific aortic valve disease, myofibroblasts and activation of the transforming growth factor-ß1 (TGF-ß1) and Wnt/ß-catenin pathways are observed in the fibrosa, the stiffer layer of the leaflet, but their association is unknown. We elucidated the roles of ß-catenin and extracellular matrix stiffness in TGF-ß1-induced myofibroblast differentiation of valve interstitial cells (VICs). METHODS AND RESULTS: TGF-ß1 induced rapid ß-catenin nuclear translocation in primary porcine aortic VICs in vitro through TGF-ß receptor I kinase. Degrading ß-catenin pharmacologically or silencing it with small interfering RNA inhibited TGF-ß1-induced myofibroblast differentiation without altering Smad2/3 activity. Conversely, increasing ß-catenin availability with Wnt3A alone did not induce differentiation. However, combining TGF-ß1 and Wnt3A caused greater myofibroblast differentiation than TGF-ß1 treatment alone. Notably, in VICs grown on collagen-coated PA gels with physiological stiffnesses, TGF-ß1-induced ß-catenin nuclear translocation and myofibroblast differentiation occurred only on matrices with fibrosa-like stiffness, but not ventricularis-like stiffness. In diseased aortic valves from pigs fed an atherogenic diet, myofibroblasts colocalized with increased protein expression of Wnt3A, ß-catenin, TGF-ß1, and phosphorylated Smad2/3 in the fibrosa. CONCLUSIONS: Myofibroblast differentiation of VICs involves matrix stiffness-dependent crosstalk between TGF-ß1 and Wnt signaling pathways and may explain in part why the stiffer fibrosa is more susceptible to disease.
Assuntos
Valva Aórtica/metabolismo , Transdiferenciação Celular , Matriz Extracelular/metabolismo , Doenças das Valvas Cardíacas/metabolismo , Miofibroblastos/metabolismo , Transdução de Sinais , Fator de Crescimento Transformador beta1/metabolismo , beta Catenina/metabolismo , Transporte Ativo do Núcleo Celular , Animais , Valva Aórtica/patologia , Células Cultivadas , Colágeno/metabolismo , Modelos Animais de Doenças , Elasticidade , Doenças das Valvas Cardíacas/patologia , Miofibroblastos/patologia , Fosforilação , Proteínas Serina-Treonina Quinases/metabolismo , Interferência de RNA , Receptor do Fator de Crescimento Transformador beta Tipo I , Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Esclerose , Proteína Smad2/metabolismo , Proteína Smad3/metabolismo , Suínos , Fatores de Tempo , Proteínas Wnt/metabolismo , Proteína Wnt3 , beta Catenina/genéticaRESUMO
Cell-substrate interactions are multifaceted, involving the integration of various physical and biochemical signals. The interactions among these microenvironmental factors cannot be facilely elucidated and quantified by conventional experimentation, and necessitate multifactorial strategies. Here we describe an approach that integrates statistical design and analysis of experiments with automated microscopy to systematically investigate the combinatorial effects of substrate-derived stimuli (substrate stiffness and matrix protein concentration) on mesenchymal stem cell (MSC) spreading, proliferation and osteogenic differentiation. C3H10T1/2 cells were grown on type I collagen- or fibronectin-coated polyacrylamide hydrogels with tunable mechanical properties. Experimental conditions, which were defined according to central composite design, consisted of specific permutations of substrate stiffness (3-144 kPa) and adhesion protein concentration (7-520 microg/mL). Spreading area, BrdU incorporation and Runx2 nuclear translocation were quantified using high-content microscopy and modeled as mathematical functions of substrate stiffness and protein concentration. The resulting response surfaces revealed distinct patterns of protein-specific, substrate stiffness-dependent modulation of MSC proliferation and differentiation, demonstrating the advantage of statistical modeling in the detection and description of higher-order cellular responses. In a broader context, this approach can be adapted to study other types of cell-material interactions and can facilitate the efficient screening and optimization of substrate properties for applications involving cell-material interfaces.
Assuntos
Resinas Acrílicas/farmacologia , Comunicação Celular/efeitos dos fármacos , Células-Tronco Mesenquimais/citologia , Microscopia/métodos , Modelos Estatísticos , Animais , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Colágeno Tipo I/farmacologia , Fibronectinas/farmacologia , Fenômenos Mecânicos/efeitos dos fármacos , Camundongos , Osteogênese/efeitos dos fármacos , Propriedades de Superfície/efeitos dos fármacosRESUMO
Adult cardiomyocytes (CM) retain little capacity to regenerate, which motivates efforts to engineer heart tissues that can emulate the functional and mechanical properties of native myocardium. Although the effects of matrix stiffness on individual CM have been explored, less attention was devoted to studies at the monolayer and the tissue level. The purpose of this study was to characterize the influence of substrate mechanical stiffness on the heart cell phenotype and functional properties. Neonatal rat heart cells were seeded onto collagen-coated polyacrylamide (PA) substrates with Young's moduli of 3, 22, 50, and 144 kPa. Collagen-coated glass coverslips without PA represented surfaces with effectively "infinite" stiffness. The local elastic modulus of native neonatal rat heart tissue was measured to range from 4.0 to 11.4 kPa (mean value of 6.8 kPa) and for native adult rat heart tissue from 11.9 to 46.2 kPa (mean value of 25.6 kPa), motivating our choice of the above PA gel stiffness. Overall, by 120 h of cultivation, the lowest stiffness PA substrates (3 kPa) exhibited the lowest excitation threshold (ET; 3.5 +/- 0.3 V/cm), increased troponin I staining (52% positively stained area) but reduced cell density, force of contraction (0.18 +/- 0.1 mN/mm(2)), and cell elongation (aspect ratio = 1.3-1.4). Higher stiffness (144 kPa) PA substrates exhibited reduced troponin I staining (30% positively stained area), increased fibroblast density (70% positively stained area), and poor electrical excitability. Intermediate stiffness PA substrates of stiffness comparable to the native adult rat myocardium (22-50 kPa) were found to be optimal for heart cell morphology and function, with superior elongation (aspect ratio > 4.3), reasonable ET (ranging from 3.95 +/- 0.8 to 4.4 +/- 0.7 V/cm), high contractile force development (ranging from 0.52 +/- 0.2 to 1.60 +/- 0.6 mN/mm(2)), and well-developed striations, all consistent with a differentiated phenotype.
Assuntos
Resinas Acrílicas/química , Técnicas de Cultura de Células/métodos , Forma Celular/efeitos dos fármacos , Colágeno/química , Módulo de Elasticidade/efeitos dos fármacos , Miócitos Cardíacos/citologia , Análise de Variância , Animais , Animais Recém-Nascidos , Contagem de Células , Sobrevivência Celular/efeitos dos fármacos , Imuno-Histoquímica , Contração Miocárdica , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Fenótipo , Ratos , Ratos Sprague-Dawley , Troponina I/metabolismo , Vimentina/metabolismoRESUMO
Synergistic interactions between cytokines underlie developmental processes fundamental to tissue and cellular engineering. However, a mechanistic understanding of the cell-specific and population-mediated effects is often lacking. In this study, we have investigated the synergistic generation of erythroid cells in response to erythropoietin (EPO) and stem cell factor (SCF). We have used a quantitative approach to determine if the effects of EPO and SCF superpose in a supra-additive fashion on the cell proliferation rate or on the death rate, suggesting a contribution from a joint cytokine effect (co-signaling). Primary mouse bone marrow hematopoietic cells and the stem cell-like FDCP-mix cell line were used to investigate the effects of EPO and SCF (individually or in combination) on erythroid output. Carboxyfluorescein diacetate succinimidyl ester (CFSE)-based cell-division tracking and mathematical modeling were used to measure cell type-specific proliferation and death rates. We observed a significant synergistic effect of EPO and SCF on the net generation of benzidine positive (erythroid) colony-forming cells, CD71++ (early erythroblasts) cells and TER-119+ (late erythroblasts and reticulocytes) cells in culture. When the observed increases in cell number were decomposed into proliferation and death rates, the cytokines were shown to act independently at different stages of erythroid development; SCF promoted the early proliferation of primitive cells, while EPO primarily promoted the survival of differentiating erythroid progenitor cells. Our analysis demonstrates that EPO and SCF have distinct and predominantly sequential effects on erythroid differentiation. This study emphasizes the necessity to separate proliferation rates from death rates to understand apparent cytokine synergies.