Porous Silk Scaffolds for Delivery of Growth Factors and Stem Cells to Enhance Bone Regeneration

Stem cell-based tissue engineering shows promise for bone regeneration and requires artificial microenvironments to enhance the survival, proliferation and differentiation of the seeded cells. Silk fibroin, as a natural protein polymer, has unique properties for tissue regeneration. The present study aimed to evaluate the influence of porous silk scaffolds on rat bone marrow stem cells (BMSCs) by lenti-GFP tracking both in vitro and in vivo in cranial bone defects. The number of cells seeded within silk scaffolds in rat cranial bone defects increased from 2 days to 2 weeks after implantation, followed by a decrease at eight weeks. Importantly, the implanted cells survived for 8 weeks in vivo and some of the cells might differentiate into endothelial cells and osteoblasts induced by the presence of VEGF and BMP-2 in the scaffolds to promote angiogenesis and osteogenesis. The results demonstrate that porous silk scaffolds provide a suitable niche to maintain long survival and function of the implanted cells for bone regeneration.     

Three Dimensional Collagen Scaffold Promotes Intrinsic Vascularisation for Tissue Engineering Applications

Here, we describe a porous 3-dimensional collagen scaffold material that supports capillary formation in vitro, and promotes vascularization when implanted in vivo. Collagen scaffolds were synthesized from type I bovine collagen and have a uniform pore size of 80 μm. In vitro, scaffolds seeded with primary human microvascular endothelial cells suspended in human fibrin gel formed CD31 positive capillary-like structures with clear lumens. In vivo, after subcutaneous implantation in mice, cell-free collagen scaffolds were vascularized by host neovessels, whilst a gradual degradation of the scaffold material occurred over 8 weeks. Collagen scaffolds, impregnated with human fibrinogen gel, were implanted subcutaneously inside a chamber enclosing the femoral vessels in rats. Angiogenic sprouts from the femoral vessels invaded throughout the scaffolds and these degraded completely after 4 weeks. Vascular volume of the resulting constructs was greater than the vascular volume of constructs from chambers implanted with fibrinogen gel alone (42.7±5.0 μL in collagen scaffold vs 22.5±2.3 μL in fibrinogen gel alone; p<0.05, n = 7). In the same model, collagen scaffolds seeded with human adipose-derived stem cells (ASCs) produced greater increases in vascular volume than did cell-free collagen scaffolds (42.9±4.0 μL in collagen scaffold with human ASCs vs 25.7±1.9 μL in collagen scaffold alone; p<0.05, n = 4). In summary, these collagen scaffolds are biocompatible and could be used to grow more robust vascularized tissue engineering grafts with improved the survival of implanted cells. Such scaffolds could also be used as an assay model for studies on angiogenesis, 3-dimensional cell culture, and delivery of growth factors and cells in vivo.     

Simple evaluation method for osteoinductive capacity of cells or scaffolds using ceramic cubes

Mesenchymal stem cells are good candidates for the clinical application of bone repair because of their osteogenic differentiation potential, but in vivo osteoinduction potential should be verified for culture expanded cells before clinical application. This study analyzed in vivo bone formation by MSCs quantitatively after implantation of MSCs planted porous biphasic ceramic cubes into athymic mice. MSCs were divided into osteogenic differentiation-induced and normal groups and also tested in vitro to evaluate the degree of differentiation into osteoblasts. The osteogenic induced group showed higher alkaline phosphatase and calcium level in vitro and corresponding higher level of bone formation in vivo compared to control group. Whereas there was no bone formation observed in fibroblast-implanted negative control group. In critical sized bone defect models, commonly used for evaluation of bone regeneration ability, it is difficult to distinguish between osteoinduction and osteoconduction, and quantitative analysis is not simple. However, this method for evaluating osteoinduction is both accurate and simple. In conclusion, the analysis of in vivo bone formation using porous ceramic cubes is a powerful and simple method for evaluating the osteoinduction ability of target cells and, furthermore, can be applied for evaluation of scaffolds for their osteoinductive properties.     

Combination of inflammation-related cytokines promotes long-term muscle stem cell expansion

Muscle stem cells (MuSCs, satellite cells) are the major contributor to muscle regeneration. Like most adult stem cells, long-term expansion of MuSCs in vitro is difficult. The in vivo muscle regeneration abilities of MuSCs are quickly lost after culturing in vitro, which prevents the potential applications of MuSCs in cell-based therapies. Here, we establish a system to serially expand MuSCs in vitro for over 20 passages by mimicking the endogenous microenvironment. We identified that the combination of four pro-inflammatory cytokines, IL-1α, IL-13, TNF-α, and IFN-γ, secreted by T cells was able to stimulate MuSC proliferation in vivo upon injury and promote serial expansion of MuSCs in vitro. The expanded MuSCs can replenish the endogenous stem cell pool and are capable of repairing multiple rounds of muscle injuries in vivo after a single transplantation. The establishment of the in vitro system provides us a powerful method to expand functional MuSCs to repair muscle injuries.     

Synergistic Actions of Hematopoietic and Mesenchymal Stem/Progenitor Cells in Vascularizing Bioengineered Tissues

Poor angiogenesis is a major road block for tissue repair. The regeneration of virtually all tissues is limited by angiogenesis, given the diffusion of nutrients, oxygen, and waste products is limited to a few hundred micrometers. We postulated that co-transplantation of hematopoietic and mesenchymal stem/progenitor cells improves angiogenesis of tissue repair and hence the outcome of regeneration. In this study, we tested this hypothesis by using bone as a model whose regeneration is impaired unless it is vascularized. Hematopoietic stem/progenitor cells (HSCs) and mesenchymal stem/progenitor cells (MSCs) were isolated from each of three healthy human bone marrow samples and reconstituted in a porous scaffold. MSCs were seeded in micropores of 3D calcium phosphate (CP) scaffolds, followed by infusion of gel-suspended CD34⁺ hematopoietic cells. Co-transplantation of CD34⁺ HSCs and CD34⁻ MSCs in microporous CP scaffolds subcutaneously in the dorsum of immunocompromized mice yielded vascularized tissue. The average vascular number of co-transplanted CD34⁺ and MSC scaffolds was substantially greater than MSC transplantation alone. Human osteocalcin was expressed in the micropores of CP scaffolds and was significantly increased upon co-transplantation of MSCs and CD34⁺ cells. Human nuclear staining revealed the engraftment of transplanted human cells in vascular endothelium upon co-transplantation of MSCs and CD34⁺ cells. Based on additional in vitro results of endothelial differentiation of CD34⁺ cells by vascular endothelial growth factor (VEGF), we adsorbed VEGF with co-transplanted CD34⁺ and MSCs in the microporous CP scaffolds in vivo, and discovered that vascular number and diameter further increased, likely owing to the promotion of endothelial differentiation of CD34⁺ cells by VEGF. Together, co-transplantation of hematopoietic and mesenchymal stem/progenitor cells may improve the regeneration of vascular dependent tissues such as bone, adipose, muscle and dermal grafts, and may have implications in the regeneration of internal organs.     

Nerve independent limb induction in axolotls

Urodele amphibians can regenerate their limbs. During limb regeneration, dermal fibroblasts are transformed into undifferentiated cells called blastema cells. These dermis–blastema cells show multipotency. Such so-called endogenous reprogramming of cell differentiation is one of the main targets of amphibian limb regeneration studies. It is well recognized that nerve presence controls the initiation of limb regeneration. Accordingly, nerve factors have been sought in amphibian limb regeneration. To investigate it, a relatively new study system called the accessory limb model (ALM) was developed. Using ALM, two signaling cascades (Fgf and Gdf5 signaling) came under focus. In the present study, Growth and differentiation factor-5 (Gdf5) application to wounded skin initiated limb regeneration responses and resulted in induction of a blastema-like structure in the absence of a nerve. However, the Gdf5-induced structure showed defects as a regeneration blastema, such as absence of detectable Prrx1 expression by in situ hybridization. The defects could be remedied by additional Fibroblasts growth factor (Fgf) inputs. These two inputs (Gdf5 and Fgfs) were sufficient to substitute for the nerve functions in the induction of limb regeneration. Indeed, Fgf2, Fgf8, and Gdf5 applications with the contralateral skin graft resulted in limb formation without nerve supply. Furthermore, acquisition of cartilage differentiation potential of dermal fibroblasts was tested in an in vivo and in vitro combination assay. Dermal fibroblasts cultured with Gdf5 were difficult to participate in cartilage formation when the cultured cells were grafted into cartilage forming region. In contrast, dermal fibroblasts cultured with Fgf2 and Fgf8 became easier to participate into cartilage formation in the same procedure. These results contribute to our understanding of molecular mechanisms of the early phase of amphibian limb regeneration.     

Recent highlights on bone stem cells: a report from Bone Stem Cells 2009, and not only…

The use of stem cells has opened new prospects for the treatment of orthopaedic conditions characterized by large bone defects. However, many issues still exist to which answers are needed before routine, large-scale application becomes possible. Bone marrow stromal cells (MSC), which are clonogenic, multipotential precursors present in the bone marrow stroma, are generally employed for bone regeneration. Stem cells with multilineage differentiation similar to MSC have also been demonstrated in adipose tissue, peripheral blood, umbilical cord and amniotic fluid. Each source presents its own advantages and drawbacks. Unfortunately, no unique surface antigen is expressed by MSC, and this hampers simple MSC enrichment from heterogeneous populations. MSC are identified through a combination of physical, morphological and functional assays. Different in vitro and in vivo models have been described for the research on bone stem cells. These models should predict the in vivo bone healing capacity of MSC and if the induced osteogenesis is similar to the physiological one. Although stem cells offer an exciting possibility of a renewable source of cells and tissues for replacement, orthopaedic applications often represent case reports whereas controlled randomized trials are still lacking. Further biological aspects of bone stem cells should be elucidated and a general consensus on the best models, protocols and proper use of scaffolds and growth factors should be achieved.     

Osteochondral Tissue Engineering In Vivo: A Comparative Study Using Layered Silk Fibroin Scaffolds from Mulberry and Nonmulberry Silkworms

The ability to treat osteochondral defects is a major clinical need. Existing polymer systems cannot address the simultaneous requirements of regenerating bone and cartilage tissues together. The challenge still lies on how to improve the integration of newly formed tissue with the surrounding tissues and the cartilage-bone interface. This study investigated the potential use of different silk fibroin scaffolds: mulberry (Bombyx mori) and non-mulberry (Antheraea mylitta) for osteochondral regeneration in vitro and in vivo. After 4 to 8 weeks of in vitro culture in chondro- or osteo-inductive media, non-mulberry constructs pre-seeded with human bone marrow stromal cells exhibited prominent areas of the neo tissue containing chondrocyte-like cells, whereas mulberry constructs pre-seeded with human bone marrow stromal cells formed bone-like nodules. In vivo investigation demonstrated neo-osteochondral tissue formed on cell-free multi-layer silk scaffolds absorbed with transforming growth factor beta 3 or recombinant human bone morphogenetic protein-2. Good bio-integration was observed between native and neo-tissue within the osteochondrol defect in patellar grooves of Wistar rats. The in vivo neo-matrix formed comprised of a mixture of collagen and glycosaminoglycans except in mulberry silk without growth factors, where a predominantly collagenous matrix was observed. Immunohistochemical assay showed stronger staining of type I and type II collagen in the constructs of mulberry and non-mulberry scaffolds with growth factors. The study opens up a new avenue of using inter-species silk fibroin blended or multi-layered scaffolds of a combination of mulberry and non-mulberry origin for the regeneration of osteochondral defects.     

Endometrial stem cells in regenerative medicine

First described in 2004, endometrial stem cells (EnSCs) are adult stem cells isolated from the endometrial tissue. EnSCs comprise of a population of epithelial stem cells, mesenchymal stem cells, and side population stem cells. When secreted in the menstrual blood, they are termed menstrual stem cells or endometrial regenerative cells. Mounting evidence suggests that EnSCs can be utilized in regenerative medicine. EnSCs can be used as immuno-modulatory agents to attenuate inflammation, are implicated in angiogenesis and vascularization during tissue regeneration, and can also be reprogrammed into induced pluripotent stem cells. Furthermore, EnSCs can be used in tissue engineering applications and there are several clinical trials currently in place to ascertain the therapeutic potential of EnSCs. This review highlights the progress made in EnSC research, describing their mesodermal, ectodermal, and endodermal potentials both in vitro and in vivo.     

Interleukin-1 Receptor-associated Kinase-4 (IRAK4) Promotes Inflammatory Osteolysis by Activating Osteoclasts and Inhibiting Formation of Foreign Body Giant Cells

Formation of foreign body giant cells (FBGCs) occurs following implantation of medical devices such as artificial joints and is implicated in implant failure associated with inflammation or microbial infection. Two major macrophage subpopulations, M1 and M2, play different roles in inflammation and wound healing, respectively. Therefore, M1/M2 polarization is crucial for the development of various inflammation-related diseases. Here, we show that FBGCs do not resorb bone but rather express M2 macrophage-like wound healing and inflammation-terminating molecules in vitro. We also found that FBGC formation was significantly inhibited by inflammatory cytokines or infection mimetics in vitro. Interleukin-1 receptor-associated kinase-4 (IRAK4) deficiency did not alter osteoclast formation in vitro, and IRAK4-deficient mice showed normal bone mineral density in vivo. However, IRAK4-deficient mice were protected from excessive osteoclastogenesis induced by IL-1β in vitro or by LPS, an infection mimetic of Gram-negative bacteria, in vivo. Furthermore, IRAK4 deficiency restored FBGC formation and expression of M2 macrophage markers inhibited by inflammatory cytokines in vitro or by LPS in vivo. Our results demonstrate that osteoclasts and FBGCs are reciprocally regulated and identify IRAK4 as a potential therapeutic target to inhibit stimulated osteoclastogenesis and rescue inhibited FBGC formation under inflammatory and infectious conditions without altering physiological bone resorption.     

Programming Stem Cells for Therapeutic Angiogenesis Using Biodegradable Polymeric Nanoparticles

Controlled vascular growth is critical for successful tissue regeneration and wound healing, as well as for treating ischemic diseases such as stroke, heart attack or peripheral arterial diseases. Direct delivery of angiogenic growth factors has the potential to stimulate new blood vessel growth, but is often associated with limitations such as lack of targeting and short half-life in vivo. Gene therapy offers an alternative approach by delivering genes encoding angiogenic factors, but often requires using virus, and is limited by safety concerns. Here we describe a recently developed strategy for stimulating vascular growth by programming stem cells to overexpress angiogenic factors in situ using biodegradable polymeric nanoparticles. Specifically our strategy utilized stem cells as delivery vehicles by taking advantage of their ability to migrate toward ischemic tissues in vivo. Using the optimized polymeric vectors, adipose-derived stem cells were modified to overexpress an angiogenic gene encoding vascular endothelial growth factor (VEGF). We described the processes for polymer synthesis, nanoparticle formation, transfecting stem cells in vitro, as well as methods for validating the efficacy of VEGF-expressing stem cells for promoting angiogenesis in a murine hindlimb ischemia model.     

The Effect of 3D Nanofibrous Scaffolds on the Chondrogenesis of Induced Pluripotent Stem Cells and Their Application in Restoration of Cartilage Defects

The discovery of induced pluripotent stem cells (iPSCs) rendered the reprogramming of terminally differentiated cells to primary stem cells with pluripotency possible and provided potential for the regeneration and restoration of cartilage defect. Chondrogenic differentiation of iPSCs is crucial for their application in cartilage tissue engineering. In this study we investigated the effect of 3D nanofibrous scaffolds on the chondrogenesis of iPSCs and articular cartilage defect restoration. Super-hydrophilic and durable mechanic polycaprolactone (PCL)/gelatin scaffolds were fabricated using two separate electrospinning processes. The morphological structure and mechanical properties of the scaffolds were characterized. The chondrogenesis of the iPSCs in vitro and the restoration of the cartilage defect was investigated using scanning electron microscopy (SEM), the Cell Counting Kit-8 (CCK-8), histological observation, RT-qPCR, and western blot analysis. iPSCs on the scaffolds expressed higher levels of chondrogenic markers than the control group. In an animal model, cartilage defects implanted with the scaffold-cell complex exhibited an enhanced gross appearance and histological improvements, higher cartilage-specific gene expression and protein levels, as well as subchondral bone regeneration. Therefore, we showed scaffolds with a 3D nanofibrous structure enhanced the chondrogenesis of iPSCs and that iPSC-containing scaffolds improved the restoration of cartilage defects to a greater degree than did scaffolds alone in vivo.     

Microencapsulated VEGF gene–modified umbilical cord mesenchymal stromal cells promote the vascularization of tissue-engineered dermis: an experimental study

Background aimsTissue-engineered dermis (TED) is thought to be the best treatment for skin defect wounds; however, lack of vascular structures in these products can cause slow vascularization or even transplant failure. We assessed the therapeutic potential of microencapsulated human umbilical cord mesenchymal stromal cells (hUCMSCs) expressing vascular endothelial growth factor (VEGF) in vascularization of TED.MethodshUCMSCs were isolated by means of enzymatic digestion and identified by means of testing biological characteristics. hUCMSCs were induced to differentiate into dermal fibroblasts in conditioned induction media. Collagen-chitosan laser drilling acellular dermal matrix (ADM) composite scaffold was prepared by means of the freeze dehydration and dehydrothermal cross-linking method. hUCMSC-derived fibroblasts were implanted on composite scaffolds to construct TED. TED with microencapsulated VEGF gene–modified hUCMSCs was then transplanted into skin defect wounds in pigs. The angiogenesis of TED at 1 week and status of wound healing at 3 weeks were observed.ResultsThe collagen-chitosan laser ADM composite has a uniform microporous structure. This composite has been used to grow hUCMSC-derived fibroblasts in vitro and to successfully construct stem cell–derived TED. Microencapsulated VEGF gene–modified hUCMSCs were prepared with the use of a sodium alginate–barium chloride one-step encapsulation technology. Seven days after the transplantation of the stem cell–derived TED and microencapsulated VEGF gene–modified hUCMSCs into the skin defect wounds on the backs of miniature pigs, the VEGF expression increased and the TED had a higher degree of vascularization. Re-epithelialization of the wound was completed after 3 weeks.ConclusionsMicroencapsulated VEGF gene–modified hUCMSCs can effectively improve the vascularization of TED and consequently the quality of wound healing.     

Electrospun PLLA nanofiber scaffolds and their use in combination with BMP-2 for reconstruction of bone defects

Introduction

Adequate migration and differentiation of mesenchymal stem cells is essential for regeneration of large bone defects. To achieve this, modern graft materials are becoming increasingly important. Among them, electrospun nanofiber scaffolds are a promising approach, because of their high physical porosity and potential to mimic the extracellular matrix (ECM).

Materials and Methods

The objective of the present study was to examine the impact of electrospun PLLA nanofiber scaffolds on bone formation in vivo, using a critical size rat calvarial defect model. In addition we analyzed whether direct incorporation of bone morphogenetic protein 2 (BMP-2) into nanofibers could enhance the osteoinductivity of the scaffolds. Two critical size calvarial defects (5 mm) were created in the parietal bones of adult male Sprague-Dawley rats. Defects were either (1) left unfilled, or treated with (2) bovine spongiosa, (3) PLLA scaffolds alone or (4) PLLA/BMP-2 scaffolds. Cranial CT-scans were taken at fixed intervals in vivo. Specimens obtained after euthanasia were processed for histology, histomorphometry and immunostaining (Osteocalcin, BMP-2 and Smad5).

Results

PLLA scaffolds were well colonized with cells after implantation, but only showed marginal ossification. PLLA/BMP-2 scaffolds showed much better bone regeneration and several ossification foci were observed throughout the defect. PLLA/BMP-2 scaffolds also stimulated significantly faster bone regeneration during the first eight weeks compared to bovine spongiosa. However, no significant differences between these two scaffolds could be observed after twelve weeks. Expression of osteogenic marker proteins in PLLA/BMP-2 scaffolds continuously increased throughout the observation period. After twelve weeks osteocalcin, BMP-2 and Smad5 were all significantly higher in the PLLA/BMP-2 group than in all other groups.

Conclusion

Electrospun PLLA nanofibers facilitate colonization of bone defects, while their use in combination with BMP-2 also increases bone regeneration in vivo and thus combines osteoconductivity of the scaffold with the ability to maintain an adequate osteogenic stimulus.     

Microporous Dermal-Mimetic Electrospun Scaffolds Pre-Seeded with Fibroblasts Promote Tissue Regeneration in Full-Thickness Skin Wounds

Electrospun scaffolds serve as promising substrates for tissue repair due to their nanofibrous architecture and amenability to tailoring of chemical composition. In this study, the regenerative potential of a microporous electrospun scaffold pre-seeded with dermal fibroblasts was evaluated. Previously we reported that a 70% collagen I and 30% poly(Ɛ-caprolactone) electrospun scaffold (70:30 col/PCL) containing 160 μm diameter pores had favorable mechanical properties, supported fibroblast infiltration and subsequent cell-mediated deposition of extracellular matrix (ECM), and promoted more rapid and effective in vivo skin regeneration when compared to scaffolds lacking micropores. In the current study we tested the hypothesis that the efficacy of the 70:30 col/PCL microporous scaffolds could be further enhanced by seeding scaffolds with dermal fibroblasts prior to implantation into skin wounds. To address this hypothesis, a Fischer 344 (F344) rat syngeneic model was employed. In vitro studies showed that dermal fibroblasts isolated from F344 rat skin were able to adhere and proliferate on 70:30 col/PCL microporous scaffolds, and the cells also filled the 160 μm pores with native ECM proteins such as collagen I and fibronectin. Additionally, scaffolds seeded with F344 fibroblasts exhibited a low rate of contraction (~14%) over a 21 day time frame. To assess regenerative potential, scaffolds with or without seeded F344 dermal fibroblasts were implanted into full thickness, critical size defects created in F344 hosts. Specifically, we compared: microporous scaffolds containing fibroblasts seeded for 4 days; scaffolds containing fibroblasts seeded for only 1 day; acellular microporous scaffolds; and a sham wound (no scaffold). Scaffolds containing fibroblasts seeded for 4 days had the best response of all treatment groups with respect to accelerated wound healing, a more normal-appearing dermal matrix structure, and hair follicle regeneration. Collectively these results suggest that microporous electrospun scaffolds pre-seeded with fibroblasts promote greater wound-healing than acellular scaffolds.     

Isolation of Basal Cells and Submucosal Gland Duct Cells from Mouse Trachea

The large airways are directly in contact with the environment and therefore susceptible to injury from toxins and infectious agents that we breath in ¹. The large airways therefore require an efficient repair mechanism to protect our bodies. This repair process occurs from stem cells in the airways and isolating these stem cells from the airways is important for understanding the mechanisms of repair and regeneration. It is also important for understanding abnormal repair that can lead to airway diseases ². The goal of this method is to isolate a novel stem cell population from the mouse tracheal submucosal gland ducts and to place these cells in in vitro and in vivo model systems to identify the mechanisms of repair and regeneration of the submucosal glands ³. This production shows methods that can be used to isolate and assay the duct and basal stem cells from the large airways ³.This will allow us to study diseases of the airway, such as cystic fibrosis, asthma and chronic obstructive pulmonary disease. Currently, there are no methods for isolation of submucosal gland duct cells and there are no in vivo models to study the regeneration of submucosal glands.     

Identification of an Effective Early Signaling Signature during Neo-Vasculogenesis In Vivo by Ex Vivo Proteomic Profiling

Therapeutic neo-vasculogenesis in vivo can be achieved by the co-transplantation of human endothelial colony-forming progenitor cells (ECFCs) with mesenchymal stem/progenitor cells (MSPCs). The underlying mechanism is not completely understood thus hampering the development of novel stem cell therapies. We hypothesized that proteomic profiling could be used to retrieve the in vivo signaling signature during the initial phase of human neo-vasculogenesis. ECFCs and MSPCs were therefore either transplanted alone or co-transplanted subcutaneously into immune deficient mice. Early cell signaling, occurring within the first 24 hours in vivo, was analyzed using antibody microarray proteomic profiling. Vessel formation and persistence were verified in parallel transplants for up to 24 weeks. Proteomic analysis revealed significant alteration of regulatory components including caspases, calcium/calmodulin-dependent protein kinase, DNA protein kinase, human ErbB2 receptor-tyrosine kinase as well as mitogen-activated protein kinases. Caspase-4 was selected from array results as one therapeutic candidate for targeting vascular network formation in vitro as well as modulating therapeutic vasculogenesis in vivo. As a proof-of-principle, caspase-4 and general caspase-blocking led to diminished endothelial network formation in vitro and significantly decreased vasculogenesis in vivo. Proteomic profiling ex vivo thus unraveled a signaling signature which can be used for target selection to modulate neo-vasculogenesis in vivo.     

Genome-wide profiling of in vivo LPS-responsive genes in splenic myeloid cells

Lipopolysaccharide (LPS), the major causative agent of bacterial sepsis, has been used by many laboratories in genome-wide expression profiling of the LPS response. However, these studies have predominantly used in vitro cultured macrophages (Macs), which may not accurately reflect the LPS response of these innate immune cells in vivo. To overcome this limitation and to identify inflammatory genes in vivo, we have profiled genome-wide expression patterns in non-lymphoid, splenic myeloid cells extracted directly from LPS-treated mice. Genes encoding factors known to be involved in mediating or regulating inflammatory processes, such as cytokines and chemokines, as well as many genes whose immunological functions are not well known, were strongly induced by LPS after 3 h or 8 h of treatment. Most of the highly LPSresponsive genes that we randomly selected from the microarray data were independently confirmed by quantitative RT-PCR, implying that our microarray data are quite reliable. When our in vivo data were compared to previously reported microarray data for in vitro LPS-treated Macs, a significant proportion (～20%) of the in vivo LPS-responsive genes defined in this study were specific to cells exposed to LPS in vivo, but a larger proportion of them (～60%) were influenced by LPS in both in vitro and in vivo settings. This result indicates that our in vivo LPS-responsive gene set includes not only previously identified in vitro LPS-responsive genes but also novel LPS-responsive genes. Both types of genes would be a valuable resource in the future for understanding inflammatory responses in vivo.