Stromal Cell Biology Research Articles

Neovascular potential of adipose‐derived stromal cells (ASCs) from diabetic patients

We explored the vascular biology of adipose-derived stromal cells (ASCs) from diabetic patients and applied these cells to a murine ischemic flap model to assess the comparative angiogenic potentials between normal and diabetic human ASCs. ASCs were obtained from diabetic patients (n = 5) and controls (n = 5). Secretion and expression of angiogenic cytokines were measured under normoxic and hypoxic condition in vitro. Conditioned media harvested from ASC cultures were assessed for their ability to stimulate human umbilical vein endothelial cell proliferation and tubulization. The control and diabetic ASCs were injected into the murine ischemic flaps, and the surviving area was measured. Diabetic adipose-derived stromal cells showed a lower level of vascular endothelial growth factor expression and cell proliferation rates than the control cells (p < 0.05). However, vascular endothelial growth factor, hepatocyte growth factor secretion, tubulogenesis, and cell proliferation in diabetic conditioned media were increased in response to hypoxic stimuli (p < 0.05), and it was similar to those of control cells. In an animal study, diabetic and normal ASCs significantly increased flap survival (p < 0.05); however, the functional difference was not found between the two groups. Diabetic ASCs were impaired in their ability to produce vascular endothelial growth factors and to induce cellular proliferation under hypoxic conditions. However, diabetic ASCs showed similar flap salvaging effect compared with controls. These findings may be important in the context of future study of autologous cell-based therapy in diabetic patients.

Evaluation of a low cost cryopreservation system on the biology of human amniotic fluid-derived mesenchymal stromal cells

BackgroundHuman amniotic-derived mesenchymal stromal cells (hAMSC) are a novel population of multipotent stem cells that have been shown to have great potential for use in regenerative medicine. However, procedures to store and preserve hAMSC for future clinical applications have not been explored extensively. MethodsIn this study, we analyzed the influence of cryopreservation, using a protocol based on freezing rate of 1°C/min, 10% dimethyl sulfoxide as cryoprotectant and a thawing rate >100°C/min, on hAMSC morphology, proliferation rates, viability, cell cycle, karyotype, immune phenotype and multilineage differentiation potential. ResultsThis study found that this cryopreservation protocol does not affect the biological properties of hAMSC. DiscussionThis shows that this protocol is a viable system for banking hAMSC, with the associated advantages that has a low cost in terms of expense, time and personnel involved and is easy to implement.

Basic Biology and Clinical Application of Multipotent Mesenchymal Stromal Cells: From Bench to Bedside

Basic Biology and Clinical Application of Multipotent Mesenchymal Stromal Cells: From Bench to Bedside

Dysregulated Bone Wound Repair and Marrow Functions in Senescence Accelerated Mice (SAMP6),

Abstract 3415 Introduction:The Senescence Accelerated-Prone mouse (SAMP6) shows normal growth followed by rapid aging, development of osteopenia and shortened lifespan, compared with control R1 mice. The bone defect has been attributed to reduced osteoblast potential of marrow stromal cells. We compared in vivo repair of tibial bone wounds. We compared in vitro hematopoiesis, irradiation sensitivity, stromal cell biology, and osteoblastogenesis with SamP6 and R1 marrow. We tested the hypothesis that SAMP6 mice have poor healing of osseous wounds and that in vitro properties of SAMP6 marrow are abnormal. Methods:As a model for spontaneous bone repair, a 2-mm unicortical wound was made in each proximal tibia. At intervals, wound diameter was measured on radiographs. Long term bone marrow cultures (LTBMCs) were established from other SAMP6 and R1 mice to measure hematopoiesis and to establish marrow stromal cell lines (MSCLs) for analysis. MSCL gene expression and sensitivity to irradiation were measured; osteoblastogenic potential was determined by culture in osteoblastogenic medium (1% FBS, 10 nM dexamathasone, 5 mM b-glycerophosphate, 50 mg/mL ascorbate-2-phosphate) and gene expression analysis for osteoblast markers alkaline phosphatase (ALP), PTH-receptor-1 (PTHR1), and Osteocalcin (OC). Results:There was a faster rate of closure of the tibial wounds in R1 than in SAMP6 mice; at day 21, wounds in R1 mice were 47% the size of those in SAMP6 mice (p = 0.032). For the first 24 weeks, SAMP6 LTBMCs had significantly greater hematopoiesis than R1, shown by more cobblestone islands (sites of stem cell activity) and by more multilineage colonies from nonadherent cells transferred to semisolid medium. After 24 weeks, however, hematopoiesis ceased in SAMP6 cultures but continued in the SAMR1 cultures for 40 weeks. There was constitutive upregulation of TGF-b, an inhibitor of hematopoiesis, in SAMP6 MSCLs, compared with R1 cells. SAMP6 MSCLs were more sensitive to radiation (Do of 1.5 ± 0.1 Gy), compared with R1 (Do of 4.0 ± 0.4 Gy; p = 0.0080). There was unexpected constitutive expression of ALP, PTHR1, and OC in SAMP6 MSCLs, compared with R1. In osteoblastogenic medium, there was greater expression of osteoblast marker genes in the SAMP6 line compared to R1. Conclusion:SAMP6 showed delayed bone wound repair. Marrow from SAMP6 mice showed shortened in vitro hematopoiesis and greater radiation sensitivity, indicative of greater oxidative stress, but the unexpected finding of constitutive expression of osteoblast genes suggest that in vivo factors are more important than innate cellular defects in marrow to account for impaired bone healing. Acknowledgments:This project was supported by NIAID U191A168021-06 and NIA R21AG034254. Disclosures:No relevant conflicts of interest to declare.

Hypoxia-Induced Angiogenesis: Good and Evil

The vascular network delivers oxygen (O(2)) and nutrients to all cells within the body. It is therefore not surprising that O(2) availability serves as a primary regulator of this complex organ. Most transcriptional responses to low O(2) are mediated by hypoxia-inducible factors (HIFs), highly conserved transcription factors that control the expression of numerous angiogenic, metabolic, and cell cycle genes. Accordingly, the HIF pathway is currently viewed as a master regulator of angiogenesis. HIF modulation could provide therapeutic benefit for a wide array of pathologies, including cancer, ischemic heart disease, peripheral artery disease, wound healing, and neovascular eye diseases. Hypoxia promotes vessel growth by upregulating multiple pro-angiogenic pathways that mediate key aspects of endothelial, stromal, and vascular support cell biology. Interestingly, recent studies show that hypoxia influences additional aspects of angiogenesis, including vessel patterning, maturation, and function. Through extensive research, the integral role of hypoxia and HIF signaling in human disease is becoming increasingly clear. Consequently, a thorough understanding of how hypoxia regulates angiogenesis through an ever-expanding number of pathways in multiple cell types will be essential for the identification of new therapeutic targets and modalities.

Molecular pathology of myelodysplastic syndromes: Biology of medullary stromal and hematopoietic cells (Review)

Myelodysplastic syndromes (MDS) have been defined as a disease entity based on clinical features and morphological findings. Despite similarities in clinical manifestations, genetic abnormalities occurring in hematopoietic cells are heterogeneous among the syndromes. However, recent investigations have revealed that there are common biological events in the bone marrow of MDS cases. Most notably, excessive apoptosis of hematopoietic cells was observed to be induced by the bone marrow microenvironment. The apoptosis was mediated by paracrine as well as autocrine factors, suggesting that medullary stromal and hematopoietic cells play a role in the pathology of disease. Pro-inflammatory cytokines, such as TNFα, in the bone marrow microenvironment are predominantly paracrine mediators of apoptosis. Regarding autocrine stimulation mechanisms, it has recently been shown that the deregulation of ribosomal protein is capable of initiating a stress response in the hematopoietic cell through a p53-mediated signaling pathway. Thus, both the stromal cells of the bone marrow microenvironment and hematopoietic cells themselves possess a common and characteristic biology in this heterogeneous disease entity.

Influence of stromal–epithelial interactions on breast cancer in vitro and in vivo

Stromal cell-secreted chemokines including CCL2 have been implicated in the primary tumor microenvironment, as mediators of tumor cell migration, proliferation, and angiogenesis. Expression of CCL2 and its principal receptor CCR2 was analyzed by RQ-PCR in primary tumor cells and breast cancer cell lines. Breast cancer cell lines (MDA-MB-231, T47D) were co-cultured directly on a monolayer of primary breast tumor and normal stromal cells, retrieved using EpCAM+ magnetic beads, and changes in expression of CCL2, CCR2, MMP11, ELK1, VIL2, and Ki67 detected by RQ-PCR. Epithelial cell migration and proliferation in response to stromal cell-secreted factors was also analyzed. In vivo, tumor xenografts were formed by co-injecting T47D cells with primary tumor stromal cells. Following establishment, tumors were harvested and digested, epithelial cells retrieved and analyzed by RQ-PCR. Whole tumor tissue was also analyzed by immunohistochemistry for CD31 and the VIL2 encoded protein Ezrin. Tumor stromal cells expressed significantly higher levels of CCL2 than normal cells, with no CCR2 expression detected. Primary epithelial cells and breast cancer cell lines expressed elevated CCL2, with relative expression of CCR2 found to be higher than the ligand. Interaction of breast cancer epithelial cells with primary tumor, but not normal stromal cells, stimulated increased expression of CCL2 (8-fold), ELK1 (6-fold), VIL2 (6-fold), and MMP11 (17-fold). Factors secreted by stromal cells, including CCL2, stimulated a significant increase in epithelial cell migration, with no effect on cell proliferation in vitro observed. In vivo, the presence of stromal cells resulted in tumors of increased volume, mediated at least in part through neoangiogenesis demonstrated by immunohistochemistry (CD31). Admixed tumor xenografts exhibited increased expression of Ki67, MMP11, VIL2, and ELK1. Elevated Ezrin protein was also detected, with increased cytoplasmic localization. The results presented highlight mechanisms through which breast cancer epithelial cells can harness stromal cell biology to support tumor progression.

Bone marrow stromal cells (bone marrow-derived multipotent mesenchymal stromal cells) for bone tissue engineering: Basic science to clinical translation

Bone tissue engineering is a promising field of regenerative medicine in which cultured cells, scaffolds, and osteogenic inductive signals are used to regenerate bone. This technology has already been used in several clinical studies and its efficacy has been reported. In this review, we focus on bone marrow stromal cells, which are the most commonly used cell source for bone tissue engineering. The nature of the cells, suitable culture conditions for bone tissue engineering, and their potential therapeutic applications are reviewed with possible caveats. Furthermore, recent advances in bone marrow stromal cell biology are discussed with reference to clinical translation.

Cytokine/Stromal Cell Networks and Lymphoid Tissue Environments

Initiation of an effective adaptive immune response against a foreign pathogen requires orchestrated encounters between lymphocytes and antigen-presenting cells. The tissues of the lymphoid system provide the ideal environment for increasing the efficiency of these encounters. Within the spleen, the mucosal-associated lymphoid tissues, and the lymph nodes, an intricate network of stromal cells, collagen fibers, and extracellular matrix exists that effectively compartmentalizes immune cells as they transit through these tissues. The stromal cells within lymphoid tissues are by no means homogenous, and it is now clear that these cells are not merely sessile bystanders during immune responses. Indeed, stromal cells within lymphoid tissues are the source of important cytokines and chemokines that guide and polarize immune cells. Here, we review the cytokines that maintain the integrity of this important stromal scaffold system within the lymphoid tissue, paying particular attention to the Lymphotoxin pathway, which is an important player in stromal cell biology. How cytokines maintain the organization of lymphoid tissues during development, in the adult animal, during inflammation and during disease will be discussed in sequence, and the clinical implications of targeting cytokines that regulate lymphoid tissue stroma will be considered.

Novel In Vitro Experimental Platform for High Throughput Analysis of the Effect of Drugs on Multiple Myeloma Cells and the Tumour Microenvironment In a Co-Culture Setting

AbstractAbstract 982Mounting evidence on the role of tumour microenvironment in supporting the growth and survival of multiple myeloma (MM) and other tumour cells makes testing of potential drug treatments in vitro in this setting almost obligatory. It is becoming evident that effective therapeutic approaches against MM must target not only MM cell viability but also the pro-survival support of the tumour cellular and non-cellular stroma. Recently developed strategies recreate the myeloma tumour microenvironment in vitro allowing for detection of myeloma cell proliferation or distribution in bone marrow compartments using cell imaging. However, there is a need for high troughput self-contained co-culture technology to facilitate differentiation in the behaviour of myeloma plasma cells from the accessory cells in the tumour microenvironment. Herein, we validate a high throughput in vitro co-culture experimental platform to analyse and measure simultaneously the effect of therapeutic agents on MM cells and the tumour stroma in co-culture. We have generated eGFP-MM cell lines (eGFP-MM1.S, eGFP-MM1.R, eGFP-U266) by lentiviral infection and clonal selection by limiting dilution and validated that eGFP-expressing cells maintain the properties of the parental cell lines. The same methodology was used to generate eGFP-K562 (myeloid malignancies), eGFP- PC3, eGFP-DU145 (both prostate cancer cell lines) and eGFP-HT29 (colon cancer cell line). We show that growth of eGFP-expressing cells can be estimated using fluorimetry (lexcitation 395/475; l emission 509) or image-based analysis in the presence of other BM cells in co-culture. The use of eGFP-MM cells also allows flow cytometry analysis of cell cycle profile and apoptosis with no significant cellular contamination from co-cultured cells such as fibroblasts, osteoclasts or stromal cells derived from bone marrow aspirates of MM patients. Additionally, we have generated and validated mCherry-expressing HS5 fibroblast cell lines as HS5 cells are commonly used to study the support by fibroblasts of proliferation and viability of MM cells. Proliferation of mCherry-HS5 cells can be evaluated using fluorimetry (l excitation 584; l emission 607) or image-based analysis in the presence of various tumour cells of haematological origin and solid tumours. The disadvantage of the MTT assay is that it is impossible to distinguish the signal of specific cell types in co-culture whereas the use of fluorescent cell lines allows us to discriminate these signals. We found that proliferation of stromal cells is differentially stimulated by different tumour cells and drug treatments. Additionally, the spatial organisation of stromal cells is also specifically altered depending on the type of tumour. These results suggest that tumour cells generate distinct signals that can affect stromal cells specifically resulting in explicit therapeutic requirements to prevent the support of the tumour microenvironment. We also found that when MM cells were seeded at low densitiy, stromal cells promote MM cell proliferation and resistance to therapeutic agents. However, when MM cells were plated at high densities and can easily enter exponential growth, tumour cells can grow independently of the presence of the stroma. However both at high and low densities, stromal cells abrogated either partially or completely the pro-apoptotic effect of therapeutic drugs. The use of eGFP-MM and mCherry HS5 cells enables application of laboratory techniques to effectively distinguish and screen the effects of drugs on the biology of myeloma cells and stromal cells in a co-culture setting that reproduces more accurately some key aspects of the tumour microenvironment. We propose the use of this experimental platform to evaluate therapeutic drugs against MM as well as other malignancies. Disclosures:No relevant conflicts of interest to declare.

Divergent Modulation of Adipose-Derived Stromal Cell Differentiation by TGF-β1 Based on Species of Derivation

Adipose-derived stromal cells hold promise for skeletal tissue engineering. However, various studies have observed that adipose-derived stromal cells differ significantly in their biology depending on species of derivation. In the following study, the authors sought to determine the species-specific response of adipose-derived stromal cells to recombinant TGF-beta1 (rTGF-beta1). Adipose-derived stromal cells were derived from mouse and human sources. Recombinant TGF-beta1 was added to culture medium (2.5 to 10 ng/ml); proliferation and osteogenic and adipogenic differentiation were assessed by standardized parameters, including cell counting, alkaline phosphatase, alizarin red, oil red O staining, and quantitative real-time polymerase chain reaction. Recombinant TGF-beta1 was found to significantly repress cellular proliferation in both mouse and human adipose-derived stromal cells (p < 0.01). Recombinant TGF-beta1 was found to significantly repress osteogenic differentiation in mouse adipose-derived stromal cells. In contrast, osteogenic differentiation of human adipose-derived stromal cells proceeded unimpeded in either the presence or the absence of rTGF-beta1. Interestingly, rTGF-beta1 induced expression of a number of osteogenic genes in human adipose-derived stromal cells, including BMP2 and BMP4. The authors' results further detail an important facet in which mouse and human adipose-derived stromal cells differ. Mouse adipose-derived stromal cell osteogenesis is completely inhibited by rTGF-beta1, whereas human adipose-derived stromal cell osteogenesis progresses in the presence of rTGF-beta1. These data highlight the importance of species of derivation in basic adipose-derived stromal cell biology. Future studies will examine in more detail the species-specific differences among adipose-derived stromal cell populations.

Organization of immunological memory by bone marrow stroma

Immunological memory is a hallmark of the adaptive immune system. Plasma cells and memory B and T cells collectively provide protective immunity and effective secondary immune responses to invading pathogens. Here, we discuss how mesenchymal stromal cells regulate immunological memory by organizing defined numbers of dedicated survival niches for plasma cells and memory T cells in the bone marrow and also, to a lesser extent, in secondary lymphoid organs. An understanding of the biology of mesenchymal stromal cells and their interaction with cells of the immune system is key to fully understanding immunological memory.

Nanoscale Topography–Induced Modulation of Fundamental Cell Behaviors of Rabbit Corneal Keratocytes, Fibroblasts, and Myofibroblasts

Keratocyte-to-myofibroblast differentiation is a key factor in corneal wound healing. The purpose of this study was to determine the influence of environmental nanoscale topography on keratocyte, fibroblast, and myofibroblast cell behavior. Primary rabbit corneal keratocytes, fibroblasts, and myofibroblasts were seeded onto planar polyurethane surfaces with six patterned areas, composed of anisotropically ordered grooves and ridges with a 400-, 800-, 1200-, 1600-, 2000-, and 4000-nm pitch (pitch = groove + ridge width). After 24 hours cells were fixed, stained, imaged, and analyzed for cell shape and orientation. For migration studies, cells on each patterned surface were imaged every 10 minutes for 12 hours, and individual cell trajectories and migration rates were calculated. Keratocytes, fibroblasts, and myofibroblasts aligned and elongated to pitch sizes larger than 1000 nm. A lower limit to the topographic feature sizes that the cells responded to was identified for all three phenotypes, with a transition zone around the 800- to 1200-nm pitch size. Fibroblasts and myofibroblasts migrated parallel to surface ridges larger than 1000 nm but lacked directional guidance on submicron and nanoscale topographic features and on planar surfaces. Keratocytes remained essentially immobile. Corneal stromal cells elongated, aligned, and migrated, differentially guided by substratum topographic features. All cell types failed to respond to topographic features approximating the dimensions of individual stromal fibers. These findings contribute to our understanding of corneal stromal cell biology in health and disease and their interaction with biomaterials and their native extracellular matrix.

Experimental basis of cord blood transplantation

Efforts are needed to enhance the efficacy of cord blood (CB) transplantation. Laboratory information set the stage for the first and subsequent CB transplants, and will be instrumental in continuing to advance the field. This paper offers a brief understanding of the current state of hematopoietic stem cell (HSC) and hematopoietic progenitor cell (HPC) biology, a look back at laboratory studies leading to the first CB transplants, and a discussion of the possible means to enhance CB transplantation. Results show that physical recovery of greater numbers of HPCs is possible after CB is collected by perfusing the placenta, but how realistic this procedure is for collection of CB to be banked is open to question. We also show that the chemokine stromal cell-derived factor-1/CXCL12 can enhance the ex vivo expansion of CB HPCs beyond that of the combination of SCF, Flt3-ligand and TPO. Advances in cytokine and stromal cell biology, and in intracellular signals mediating the effects of cytokines/stromal cells should be considered in the context of future efforts to enhance functional activities of donor CB HSCs and HPCs and the microenvironmental niche of the recipient, which is required for acceptance and nurturing these HSCs/HPCs.

Cord Blood—An Alternative Source for Bone Regeneration

Bone regeneration is one of the best investigated pathways in mesenchymal stromal cell (MSC) biology. Therefore strong efforts have been made to introduce tissue engineering and cell therapeutics as an alternative treatment option for patients with bone defects. This review of the literature gives an overview of MSC biology aiming for clinical application including advantages but also specific challenges and problems which are associated with cord blood derived stromal cell (CB-MSC) as a source for bone regeneration. The use of postnatal CB-MSC is ethically uncomplicated and requires no invasive harvesting procedure. Moreover, most data document a high osteogenic potential of CB-MCS and also low immunoreactivity compared with other MSC types. The expression profile of CB-MSC during osteogenic differentiation shows similarities to that of other MSC types. Within the umbilical cord different MSC types have been characterized which are potent to differentiate into osteoblasts. In contrast to a large number of in vitro investigations there are only few in vivo studies available so far.

Genomics Analysis of Freshly-Purified Human Marrow Stromal Cells (MSCs): Comparison with Hematopoietic Lineage Cells and Culture-Expanded MSCs.

Gene and antigen expression profile of bone marrow MSCs in culture has been investigated, but these characteristics of MSCs in vivo are poorly understood. In this study we used microarrays to compare MSCs freshly-purified from human bone marrow aspirates (f-MSCs) with donor-matched hematopoietic lineage cells (HLCs) and with standard culture-expanded MSCs (e-MSCs). Following enrichment with Anti-Fibroblast MicroBeads, f-MSCs were purified by cell sorting as CD45lowCD235a-LNGFR+ [1] and HLCs as CD45+CD235a+LNGFR-. RNA was extracted, amplified and hybridized to an Affimetrix U133 Plus 2.0 chip. Raw fluorescence data were normalised to GAPDH for direct comparison with qPCR. Duplicate hybridizations were used to define 3 relative zones of sensitivity and select appropriate thresholds for significant changes in expression. The microarray data were validated by qPCR using several mesenchymal lineage genes (including Sox9 and Runx2) and by flow cytometry for several known MSC surface markers (including CD73, CD90, CD146, CD10, CD63 and CD130). Expression data were further searched for gene ontology using a custom-made, Perl-based program. Compared to HLCs, f-MSCs over-expressed genes encoding extracellular matrix proteins (including versican, fibronectin and collagen I), stromal cytokines/chemokines (including SDF-1, IL-7 and CXCL10), and molecules associated with cell junctions and communication with endothelium (including connexin 43, VCAM-1 and angiopoietin-1). Osteogenesis-related genes (BMPs and their receptors, alkaline phosphatase, osteonectin, osteocalcin, osteomodulin and others) were more abundant in f-MSCs than in HLCs. Some mature fat tissue products were also quite abundant in f-MSCs (particularly lipoprotein lipase and adipsin), suggesting the presence of cells committed to adipogenesis. Of note, the expression of stroma- and fat-related transcripts was significantly reduced in e-MSCs compared to f-MSCs. Novel surface antigens of f-MSCs identified by microarray, Junctional Adhesion Molecule 2 (normally expressed on vascular endothelial cells), Leptin receptor (implicated in fat and bone metabolism), and CD81 (tetraspanin family adhesion molecule) were confirmed at the protein level by 6-colour flow cytometry. Altogether, this work confirmed the CD45lowCD235a-LNGFR+ phenotype f-MSCs in human bone marrow, revealed a unique expression profile of f-MSCs in vivo and described new surface markers of f-MSCs. These data have implications for our understanding of the in vivo biology of non-hematopoietic bone marrow stromal cells and their role in hematopoiesis and skeletal remodelling in health and disease.

Homing efficiency and hematopoietic reconstitution of bone marrow-derived stroma cells expanded by recombinant human macrophage-colony stimulating factor in vitro

The increasing recognition of the properties of marrow stromal cells has spawned a major switch in our perception of their nature and the potential therapeutic applications that have been envisioned and implemented. Yet, several aspects of bone marrow stromal cell biology remain in question. This report describes the ability of recombinant human macrophage colony-stimulating factor (rhM-CSF) to maintain proliferation and differentiation of bone marrow stromal cells ex vivo. Our results demonstrated that M-CSF was essential for proliferation and differentiation of bone marrow-derived stromal cells and exerted its effects in a dose-dependent manner. The number of colony-forming unit (CFU) fibroblasts increased by 25% after incubation with rhM-CSF. In vitro expanded bone marrow stromal cells were easy to passage and differentiated to adipocyte and chondroblast cells under appropriate culture conditions. Furthermore, these expanded stromal cells to support CD34+ hematopoietic stem cells, as demonstrated by their ability to form CFU-Mix, burst-forming units-erythroid, and CFU-granulocyte macrophage colonies after 3 weeks of culture. The homing efficiency of in vitro expanded or fresh isolated bone marrow-derived stromal cells, which were labeled with carboxy fluorescein diacetate succinimidyl ester, to bone marrow was also investigated. Homing assays demonstrated that freshly isolated CD45+-depleted bone marrow cells were able to home to bone marrow in a dose-dependent manner, although some cells were found in the spleen, liver, and lung. However, their ability to home was dramatically reduced with culture time and was completely lost after five to seven passages in vitro. Animal studies showed that freshly isolated or rhM-CSF-induced bone marrow stromal cells promoted hematopoietic reconstitution in lethally irradiated mice. The ability to easily expand human stromal cells, which support survival and proliferation of CD34+ cells, has many important clinical applications for hematopoietic disorders.

Vertebrate Thymus and the Neurotrophin System

An immunomodulary role has been proposed for growth factors included in the family of neurotrophins. This is supported by the presence of both neurotrophins and neurotrophin receptors in the immune organs and some immunocompetent cells, the in vitro and in vivo effects of the neurotrophins on the immune cells, and the structural changes of lymphoid organs in mice deficient in neurotrophins and their receptors. The current data strongly indicate that neurotrophins regulate the biology of thymic stromal cells and T cells, including survival, and are involved in the thymic organogenesis. This review compiles the available data about the occurrence and distribution of neurotrophins and their signaling receptors (Trk proteins and p75(NTR)) in the vertebrate thymus and the possible contribution of these molecules to the thymic microenvironment and, therefore, to the T cells differentiation.

Spatial, a gene expressed in thymic stromal cells, depends on three-dimensional thymus organization for its expression.

Although the importance of thymic stroma in thymopoiesis has been recognized, the underlying molecular details regarding stromal cell biology remain obscure. To study this area, we have cloned genes expressed in thymic stromal cells. Spatial is alternatively spliced to generate two mRNAs in thymus and lymph node (LN) but it is not expressed in the spleen. In mouse embryos, the short form begins expression at day 10 while the long form is not detected until day 12. Both mRNAs encode proline rich proteins and their closest homology is to homeobox and POU domain transcription factors. Spatial is not expressed in thymocytes, but it is expressed in 2-deoxyguanosine-treated day 14 fetal thymic organ culture (FTOC) and in reaggregated FTOC. These data suggest that a normal three-dimensional organization of stromal cells is required for Spatial expression. An antiserum raised against a C-terminal peptide detected proteins of 38 and 32 kDa in Western blots of total thymus proteins. In frozen thymus sections, subcapsular epithelial cells were stained with the anti-Spatial antiserum. Paracortical subcapsular cells of unknown function were also stained in the LN. Both forms of Spatial fused to the green fluorescent protein (GFP) localize to the nucleus in transfected cells.

Diseases of bone and the stromal cell lineage.

Diseases of bone and the stromal cell lineage.