Multipotent mesenchymal stromal cells are sensitive to thermic stress - potential implications for therapeutic hyperthermia.

Purpose: Hyperthermia demonstrated clinical efficacy in multimodal cancer treatment. Multipotent mesenchymal stromal cells (MSCs) as part of the tumor-supporting stroma modulate tumor response and tissue regeneration after hyperthermia. We aimed to investigate the effects of hyperthermia on the survival, stem cell characteristics and heat shock expression of human MSCs.Materials and methods: Human MSCs and normal human dermal fibroblasts (NHDFs) were exposed to temperatures between 37 °C and 44 °C for 60 min, and hyperthermic sensitivity was examined by clonogenicity, proliferation and viability assays. The influence of 42 °C hyperthermia on the MSCs' adhesion potential, migratory capacity, surface marker expression and multi-lineage differentiation capability was investigated. Cell cycle distribution, apoptosis and senescence after 42 °C hyperthermia were determined by flow cytometry and ß-galactosidase staining. Heat shock protein expression was determined by Western Blots.Results: MSCs exhibited decreased clonogenic survival after 40 °C and 42 °C hyperthermia compared to NHDFs, while proliferative activity and viability were comparable after hyperthermia up to 44 °C. MSC adhesion was reduced after 42 °C hyperthermia, while the characteristic surface marker expression and the migratory ability remained unaffected in 42 °C hyperthermia-exposed MSCs. 42 °C hyperthermia diminished the adipogenic differential potential of all tested MSC samples. A pronounced G2/M arrest was found after 42 °C hyperthermia and was associated with increased apoptosis and senescence levels in MSCs. MSCs exhibited slightly lower heat shock protein levels compared to NHDFs.Conclusion: Human MSCs exhibit a thermosensitive phenotype which reduced the multipotent cells' regenerative abilities, resulting in impaired tissue regeneration after hyperthermia treatment or thermal injuries. On the other hand, tumor-associated MSCs may be efficiently targeted by hyperthermia treatment.

The current understanding of mesenchymal stem cells as potential attenuators of chemotherapy-induced toxicity.

Chemotherapeutic agents are part of the standard treatment algorithms for many malignancies; however, their application and dosage are limited by their toxic effects to normal tissues. Chemotherapy-induced toxicities can be long-lasting and may be incompletely reversible; therefore, causative therapies for chemotherapy-dependent side effects are needed, especially considering the increasing survival rates of treated cancer patients. Mesenchymal stem cells (MSCs) have been shown to exhibit regenerative abilities for various forms of tissue damage. Preclinical data suggest that MSCs may also help to alleviate tissue lesions caused by chemotherapeutic agents, mainly by establishing a protective microenvironment for functional cells. Due to the systemic administration of most anticancer agents, the effects of these drugs on the MSCs themselves are of crucial importance to use stem cell-based approaches for the treatment of chemotherapy-induced tissue toxicities. Here, we present a concise review of the published data regarding the influence of various classes of chemotherapeutic agents on the survival, stem cell characteristics and physiological functions of MSCs. Molecular mechanisms underlying the effects are outlined, and resulting challenges of MSC-based treatments for chemotherapy-induced tissue injuries are discussed.

Microcavity arrays as an in vitro model system of the bone marrow niche for hematopoietic stem cells.

In previous studies human mesenchymal stromal cells (MSCs) maintained the "stemness" of human hematopoietic progenitor cells (HPCs) through direct cell-cell contact in two-dimensional co-culture systems. We establish a three-dimensional (3D) co-culture system based on a custom-made chip, the 3(D)-KITChip, as an in vitro model system of the human hematopoietic stem cell niche. This array of up to 625 microcavities, with 300 µm size in each orientation, was inserted into a microfluidic bioreactor. The microcavities of the 3(D)-KITChip were inoculated with human bone marrow MSCs together with umbilical cord blood HPCs. MSCs used the microcavities as a scaffold to build a complex 3D mesh. HPCs were distributed three-dimensionally inside this MSC network and formed ß-catenin- and N-cadherin-based intercellular junctions to the surrounding MSCs. Using RT(2)-PCR and western blots, we demonstrate that a proportion of HPCs maintained the expression of CD34 throughout a culture period of 14 days. In colony-forming unit assays, the hematopoietic stem cell plasticity remained similar after 14 days of bioreactor co-culture, whereas monolayer co-cultures showed increasing signs of HPC differentiation and loss of stemness. These data support the notion that the 3D microenvironment created within the microcavity array preserves vital stem cell functions of HPCs more efficiently than conventional co-culture systems.

Standardization of Good Manufacturing Practice-compliant production of bone marrow-derived human mesenchymal stromal cells for immunotherapeutic applications.

BACKGROUND AIMS: Human mesenchymal stem or stromal cells (MSCs) represent a potential resource not only for regenerative medicine but also for immunomodulatory cell therapies. The application of different MSC culture protocols has significantly hampered the comparability of experimental and clinical data from different laboratories and has posed a major obstacle for multicenter clinical trials. Manufacturing of cell products for clinical application in the European Community must be conducted in compliance with Good Manufacturing Practice and requires a manufacturing license. In Germany, the Paul-Ehrlich-Institut as the Federal Authority for Vaccines and Biomedicines is critically involved in the approval process. METHODS: This report summarizes a consensus meeting between researchers, clinicians and regulatory experts on standard quality requirements for MSC production. RESULTS: The strategy for quality control testing depends on the product's cell composition, the manufacturing process and the indication and target patient population. Important quality criteria in this sense are, among others, the immunophenotype of the cells, composition of the culture medium and the risk for malignant transformation, as well as aging and the immunosuppressive potential of the manufactured MSCs. CONCLUSIONS: This position paper intends to provide relevant information to interested parties regarding these criteria to foster the development of scientifically valid and harmonized quality standards and to support approval of MSC-based investigational medicinal products.

Plerixafor induces the rapid and transient release of stromal cell-derived factor-1 alpha from human mesenchymal stromal cells and influences the migration behavior of human hematopoietic progenitor cells.

The interaction between the stromal cell-derived factor-1 alpha (SDF-1α, CXCL12) and its chemokine receptor CXCR4 has been reported to regulate stem cell migration, mobilization and homing. The CXCR4 antagonist plerixafor is highly efficient in mobilizing hematopoietic progenitor cells (HPCs). However, the precise regulatory mechanisms governing the CXCR4/SDF-1α axis between the bone marrow niche and HPCs remain unclear. In this study, we quantify the impact of plerixafor on the interaction between human bone marrow derived mesenchymal stromal cells (MSCs) and human CD34+ HPCs. An assessment of SDF-1α levels in the supernatant of MSC cultures revealed that exposure to plerixafor led to a transient increase but had no long-term effect. In Transwell experiments, we observed that the addition of SDF-1α significantly stimulated HPC migration; this stimulation was almost completely antagonized by the addition of plerixafor, confirming the direct impact of the CXCR4/SDF-1α interaction on the migration capacity of HPCs. We also developed a new microstructural niche model to determine the chemotactic sensitivity of HPCs. Time-lapse microscopy demonstrated that HPCs migrated actively along an SDF-1α gradient within the microchannels and the quantitative assessment of the required minimum gradient initiating this chemotaxis revealed a surprisingly high sensitivity of HPCs. These data demonstrate the fine-tuned balance of the CXCR4/SDF-1α axis and the synergistic effects of plerixafor on HPCs and MSCs, which most likely represent the key mechanisms for the consecutive mobilization of HPCs from the bone marrow niche into the circulating blood.

Mesenchymal stem cells are sensitive to treatment with kinase inhibitors and ionizing radiation.

INTRODUCTION: Mesenchymal stem cells (MSCs) can regenerate damaged tissues and may therefore be of importance for normal tissue repair after cancer treatment. Small molecule receptor kinase inhibitors (RKIs) have recently been introduced into cancer treatment. However, the influence of these drugs-particularly in combination with radiotherapy-on the survival of MSCs is largely unknown. METHODS: The sensitivity of human primary MSCs from healthy volunteers and primary human fibroblast cells to small molecule kinase inhibitors of the vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF) and transforming growth factor ß (TGFß) receptors, as well to inhibitors of c-Kit, was examined in combination with ionizing radiation (IR); cell survival and proliferation were assessed. Expression patterns of different kinase receptors and ligands were investigated using gene arrays. RESULTS: MSCs were highly sensitive to the tyrosine kinase inhibitors SU14816 (imatinib) and SU11657 (sunitinib), but showed only moderate sensitivity to the selective TGFß receptor 1 inhibitor LY2109761. Primary adult human fibroblasts were comparably resistant to all three inhibitors. The addition of IR had an additive or supra-additive effect in the MSCs, but this was not the case for differentiated fibroblasts. Proliferation was markedly reduced in MSCs following kinase inhibition, both with and without IR. Gene expression analysis revealed high levels of the PDGF α and ß receptors, and lower levels of the TGFß receptor 2 and Abl kinase. IR did not alter the expression of kinase receptors or their respective ligands in either MSCs or adult fibroblasts. CONCLUSION: These data show that MSCs are highly sensitive to RKIs and combination treatments incorporating IR. Expression analyses suggest that high levels of PDGF receptors may contribute to this effect.

Functional potentials of human hematopoietic progenitor cells are maintained by mesenchymal stromal cells and not impaired by plerixafor.

BACKGROUND AIMS: Mesenchymal stromal cells (MSCs) resemble an essential component of the bone marrow niche for maintenance of stemness of hematopoietic progenitor cells (HPCs). Perturbation of the C-X-C chemokine receptor type 4 (CXCR4)/stromal cell-derived factor-1α (SDF-1α) axis by plerixafor (AMD3100) mobilizes HPCs from their niche; however, little is known about how plerixafor affects interaction of HPCs and MSCs in vitro. METHODS: We monitored cell division kinetics, surface expression of CD34 and CXCR4, migration behavior and colony-forming frequency of HPCs on co-culture with MSCs either with or without exposure to plerixafor. RESULTS: Co-culture with MSCs significantly accelerated cell division kinetics of HPCs. Despite this, the proportion of CD34⁺ cells was significantly increased on co-culture, whereas the expression of CXCR4 was reduced. In addition, co-culture with MSCs led to significantly higher colony-forming capacity and enhanced migration rate of HPCs compared with mono-culture conditions. The composition of MSC sub-populations-and conversely their hematopoiesis supportive functions-may be influenced by culture conditions. We compared the stromal function of MSCs isolated with three different culture media. Overall, the supporting potentials of these MSC preparations were quite similar. Perturbation by the CXCR4-antagonist plerixafor reduced the cell division kinetics of HPCs on co-culture with MSCs. However, the progenitor cell potential of the HPCs as reflected by colony-forming capacity was not affected by plerixafor. CONCLUSIONS: These results support the notion that the CXCR4/SDF-1α axis is critical for HPC-MSC interaction with regard to migration, adhesion and regulation of proliferation but not for maintenance of primitive progenitor cells.

Characterization of hematopoietic stem cell subsets from patients with multiple myeloma after mobilization with plerixafor.

BACKGROUND AIMS: Previous studies have demonstrated that the combination of granulocyte-colony-stimulating factor (G-CSF) + plerixafor is more efficient in mobilizing CD34(+) hematopoietic stem cells (HSC) into the peripheral blood than G-CSF alone. In this study we analyzed the impact of adding plerixafor to G-CSF upon the mobilization of different HSC subsets. METHODS: We characterized the immunophenotype of HSC subsets isolated from the peripheral blood of eight patients with multiple myeloma (MM) before and after treatment with plerixafor. All patients were supposed to collect stem cells prior to high-dose chemotherapy and consecutive autologous stem cell transplantation, and therefore received front-line mobilization with 4 days of G-CSF followed by a single dose of plerixafor. Samples of peripheral blood were analyzed comparatively by flow cytometry directly before and 12 h after administration of plerixafor. RESULTS: The number of aldehyde dehydrogenase (ALDH)(bright) and CD34(+) cells was significantly higher after plerixafor treatment (1.2-5.0 and 1.5-6.0 times; both P < 0.01) and an enrichment of the very primitive CD34(+) CD38(-) and ALDH(bright) CD34(+) CD38(-) HSC subsets was detectable. Additionally, two distinct ALDH(+) subsets could be clearly distinguished. The small ALDH(high) subset showed a higher number of CD34(+) CD38(-) cells in contrast to the total ALDH(bright) subpopulation and probably represented a very primitive subpopulation of HSC. CONCLUSIONS: A combined staining of ALDH, CD34 and CD38 might represent a powerful tool for the identification of a very rare and primitive hematopoietic stem cell subset. The addition of plerixafor mobilized not only more CD34(+) cells but was also able to increase the proportion of more primitive stem cell subsets.

Co-culture with mesenchymal stromal cells increases proliferation and maintenance of haematopoietic progenitor cells.

Mesenchymal stromal cells (MSC) have been suggested to provide a suitable cellular environment for in vitro expansion of haematopoietic stem and progenitor cells (HPC) from umbilical cord blood. In this study, we have simultaneously analysed the cell division history and immunophenotypic differentiation of HPC by using cell division tracking with carboxyfluorescein diacetate N-succinimidyl ester (CFSE). Co-culture with MSC greatly enhanced proliferation of human HPC, especially of the more primitive CD34(+)CD38(-) fraction. Without co-culture CD34 and CD133 expressions decreased after several cell divisions, whereas CD38 expression was up-regulated after some cell divisions and then diminished in fast proliferating cells. Co-culture with MSC maintained a primitive immunophenotype (CD34(+), CD133(+) and CD38(-)) for more population doublings, whereas up-regulation of differentiation markers (CD13, CD45 and CD56) in HPC was delayed to higher numbers of cell divisions. Especially MSC of early cell passages maintained CD34 expression in HPC over more cell divisions, whereas MSC of higher passages further enhanced their proliferation rate. Inhibition of mitogen-activated protein kinase 1 (MAPK1) impaired proliferation and differentiation of HPC, but not maintenance of long-term culture initiating cells. siRNA knockdown of N-cadherin and VCAM1 in feeder layer cells increased the fraction of slow dividing HPC, whereas knockdown of integrin beta 1 (ITGB1) and CD44 impaired their differentiation. In conclusion, MSC support proliferation as well as self-renewal of HPC with primitive immunophenotype. The use of early passages of MSC and genetic manipulation of proteins involved in HPC-MSC interaction might further enhance cord blood expansion on MSC.

Induction of CD4+ T-cell anergy and apoptosis by activated human B cells.

B cells are well-known mediators of humoral immunity and serve as costimulators in the generation of T cell-mediated responses. In several mouse models, however, it was observed that B cells can also down-regulate immune reactions, suggesting a dual role for B cells. Due to this discrepancy and so far limited data, we directly tested the effects of primary human B cells on activated CD4(+) T helper cells in vitro. We found that under optimal costimulation large, activated CD25(+) B cells but not small CD25(-) B cells induced temporary T-cell anergy, determined by cell division arrest and down-regulation of cytokine production. In addition, large CD25(+) B cells directly induced CD95-independent apoptosis in a subpopulation of activated T cells. Suppression required direct B-T-cell contact and was not transferable from T to T cell, excluding potential involvement of regulatory T cells. Moreover, inhibitory effects involved an IL-2-dependent mechanism, since decreasing concentrations of IL-2 led to a shift from inhibitory toward costimulatory effects triggered by B cells. We conclude that activated CD25(+) B cells are able to costimulate or down-regulate T-cell responses, depending on activation status and environmental conditions that might also influence their pathophysiological impact.

Effect of Increased Lactate Dehydrogenase A Activity and Aerobic Glycolysis on the Proinflammatory Profile of Autoimmune CD8+ T Cells in Rheumatoid Arthritis.

OBJECTIVE: CD8+ T cells contribute to rheumatoid arthritis (RA) by releasing proinflammatory and cytolytic mediators, even in a challenging hypoxic and nutrient-poor microenvironment such as the synovial membrane. This study was undertaken to explore the mechanisms through which CD8+ T cells meet their metabolic demands in the blood and synovial membrane of patients with RA. METHODS: Purified blood CD8+ T cells from patients with RA, patients with psoriatic arthritis (PsA), and patients with spondyloarthritis (SpA), as well as healthy control subjects, and CD8+ T cells from RA synovial membrane were stimulated in medium containing 13 C-labeled metabolic substrates in the presence or absence of metabolic inhibitors, under conditions of normoxia or hypoxia. The production of metabolic intermediates was quantified by 1 H-nuclear magnetic resonance. The expression of metabolic enzymes, transcription factors, and immune effector molecules was assessed at both the messenger RNA (mRNA) and protein levels. CD8+ T cell functional studies were performed. RESULTS: RA blood CD8+ T cells met their metabolic demands through aerobic glycolysis, production of uniformly 13 C-enriched lactate in the RA blood (2.6 to 3.7-fold higher than in patients with SpA, patients with PsA, and healthy controls; P < 0.01), and induction of glutaminolysis. Overexpression of Warburg effect-linked enzymes in all RA CD8+ T cell subsets maintained this metabolic profile, conferring to the cells the capacity to proliferate under hypoxia and low-glucose conditions. In all RA CD8+ T cell subsets, lactate dehydrogenase A (LDHA) was overexpressed at the mRNA level (P < 0.03 versus controls; n = 6 per group) and protein level (P < 0.05 versus controls; n = 17 RA patients, n = 9 controls). In RA blood, inhibition of LDHA with FX11 led to reductions in lipogenesis, migration and proliferation of CD8+ T cells, and CD8+ T cell effector functions, while production of reactive oxygen species was increased by 1.5-fold (P < 0.03 versus controls). Following inhibition of LDHA with FX11, RA CD8+ T cells lost their capacity to induce healthy B cells to develop a proinflammatory phenotype. Similar metabolic alterations were observed in RA CD8+ T cells from the synovial membrane. CONCLUSION: Remodeling glucose and glutamine metabolism in RA CD8+ T cells by targeting LDHA activity can reduce the deleterious inflammatory and cytolytic contributions of these cells to the development of autoimmunity.

A Microcavity Array-Based 3D Model System of the Hematopoietic Stem Cell Niche.

Despite huge advances in recent years, the interaction between hematopoietic stem and progenitor cells (HSPCs) and their niches in the bone marrow is still far from being fully understood. One reason is that hematopoiesis is a multi-step maturation process leading to HSPC heterogeneity. Subpopulations of HSPCs can be identified by clonogenic assays or in serial transplantation experiments in mice following sublethal irradiation, but it is very complex to reproduce or even maintain stem cell plasticity in vitro. Advanced model systems have been developed that allow to precisely control and analyze key components of the physiologic microenvironment for not only fundamental research purposes but, as a long-term goal, also for clinical applications. In this chapter, we describe our approach of building an artificial hematopoietic stem cell niche in the form of polymer film-based microcavities with a diameter of 300 µm and a depth of up to 300 µm and arranged in a 634-cavity array. The polymer films are provided with 3 µm pores and thus allow perfusion of the culture medium. The microcavity arrays can be inserted into a microbioreactor where a closed circulation loop can be tightly controlled with regard to medium flow and gas supply. The microcavity arrays were used for a three-dimensional (3D) co-culture of MSCs and HSPCs in a defined ratio over a time period of up to 21 days. With this setup, it could be demonstrated that the HSPCs maintained their stem cell characteristics more efficiently as compared to conventional monolayer co-culture controls.

Human mesenchymal stem cells are resistant to UV-B irradiation.

Albeit being an effective therapy for various cutaneous conditions, UV-B irradiation can cause severe skin damage. While multipotent mesenchymal stem cells (MSCs) may aid the regeneration of UV-B-induced skin injuries, the influence of UV-B irradiation on MSCs remains widely unknown. Here, we show that human MSCs are relatively resistant to UV-B irradiation compared to dermal fibroblasts. MSCs exhibited higher clonogenic survival, proliferative activity and viability than dermal fibroblasts after exposure to UV-B irradiation. Cellular adhesion, morphology and expression of characteristic surface marker patterns remained largely unaffected in UV-irradiated MSCs. The differentiation ability along the adipogenic, osteogenic and chondrogenic lineages was preserved after UV-B treatment. However, UV-B radiation resulted in a reduced ability of MSCs and dermal fibroblasts to migrate. MSCs exhibited low apoptosis rates after UV-B irradiation and repaired UV-B-induced cyclobutane pyrimidine dimers more efficiently than dermal fibroblasts. UV-B irradiation led to prolonged p53 protein stability and increased p21 protein expression resulting in a prolonged G2 arrest and senescence induction in MSCs. The observed resistance may contribute to the ability of these multipotent cells to aid the regeneration of UV-B-induced skin injuries.

Mesenchymal stem cells preserve their stem cell traits after exposure to antimetabolite chemotherapy.

BACKGROUND: Mesenchymal stem cells (MSCs) participate in the regeneration of tissue lesions induced by antimetabolite chemotherapy; however, the influence of this class of anti-cancer compounds on the stem cells remains largely unknown. METHODS: The survival of MSCs after exposure to 5-fluorouracil (5-FU) and gemcitabine was measured by viability and clonogenic assays. MSC morphology, surface marker expression, adhesion potential, cellular velocity and differentiation potential were determined after antimetabolite treatment. Cell cycle distribution and apoptosis were assessed using flow cytometry, and senescence induction was evaluated by beta-galactosidase staining. Gene expression arrays were used to analyze the expression of enzymes involved in DNA metabolism and multidrug resistance. RESULTS: Here, we show that human primary bone marrow MSCs are relatively resistant to treatment with the widely used antimetabolite drugs 5-FU and gemcitabine. The stem cells were able to largely retain their functional abilities and defining stem cell traits after antimetabolite exposure. MSCs surface markers were found stably expressed, and the characteristic multi-lineage differentiation potential was maintained irrespective of 5-FU or gemcitabine treatment. High expression levels of enzymes involved in DNA metabolism and multidrug resistance transporters may contribute to the resistance to antimetabolite chemotherapy. DISCUSSION: The observed resistance and functional integrity may form the basis for further investigations of MSCs as a potential therapy for antimetabolite-induced tissue damage.

Adhesion of human hematopoietic progenitor cells to mesenchymal stromal cells involves CD44.

BACKGROUND: Direct cell-cell contact between hematopoietic progenitor cells (HPC) and their cellular microenvironment is essential for maintenance of 'stemness'. We have previously demonstrated that a feeder layer of human mesenchymal stromal cells (MSC) could provide a surrogate model as a niche for human HPC. Maintenance of long-term culture-initiating cells was significantly lower on fibroblasts. METHODS: Adhesion of HPC to MSC was further analyzed using our recently described adhesion assay based on gravitational force upon inversion and in combination with specific antibodies against CD44. RESULTS: Adhesion of KG1a and CD34+ cells was significantly reduced by administration of a monoclonal CD44 antibody and for KG1a to a greater extent than for CD34+ cells. Interaction of HPC and MSC was further analyzed by laser scanning confocal microscopy. CD44 was located on the uropod of CD34+ cells at the site of contact with MSC and both cell types were interwoven by a network of fibronectin. CONCLUSION: Various adhesion proteins, including CD44, are involved in the contact of human HPC and human MSC and further analysis of the relative significance and interaction of these proteins will be crucial for the understanding of the mechanism of this specific cell-cell interaction.

The Stromal Activity of Mesenchymal Stromal Cells.

SUMMARY: The mechanism that regulates self-renewal and differentiation of hematopoietic stem cells (HSC) is a central question in stem cell biology that might ultimately lead to reliable protocols for in vitro expansion of HSC. Cellular fate is governed by cell-cell interaction with the microenvironment in the bone marrow, the stem cell niche. Mesenchymal stromal cells (MSC) are precursors of the cellular components, and they secrete extracellular matrix proteins of the bone marrow stroma. Therefore, MSC feeder layer might provide a suitable in vitro model system for the stem cell niche. In vitro assays demonstrate that MSC maintain the stem cell function of HSC and that MSC from bone marrow have a higher hematopoiesis supportive activity than MSC from adipose tissue. Co-cultivation with MSC might pave the way for expansion of long-term repopulating HSC, and various clinical trials indicate that co-transplantation of HSC and MSC might enhance engraftment. Thus, MSC are promising tools to elucidate the underlying mechanism of the cellular microenvironment. The large variety of preparative protocols for isolation and cultivation of MSC affects their stromal activity. Standardized isolation methods and molecular characterization of MSC are of utmost importance for reproducible isolation of hematopoiesis supportive stromal cells and for their potential clinical application.

Primitive and committed human hematopoietic progenitor cells interact with primary murine neural cells and are induced to undergo self-renewing cell divisions.

OBJECTIVE: Studies in animal models have indicated that hematopoietic progenitor cells (HPC) migrate and home to the central nervous system and might acquire neural features under specific circumstances. The interaction between HPC and the neural environment and the functional effect on hematopoiesis have not yet been defined. METHODS: CD34(+)133(+) cells from mobilized peripheral blood were cocultured with primary murine neurons or astrocytes. Chemotaxis and adhesive interactions were studied by applying beta(1)- and beta(2)-integrin function-blocking anibodies. The impact of neural feeder layers on integrin expression of HPC and the presence of appropriate adhesion ligands on neural cells were determined by immunostaining and flow cytometry. The hematopoietic long-term fate was monitored by time-lapse microscopy of individual cell-division history followed by long-term culture-initiating cell (LTC-IC) and colony-forming cell (CFC) assays. Neural differentiation was assessed by immunostaining against specific neuronal and glial antigens. RESULTS: The 23.0% +/- 4.9% of HPC showed stromal cell-derived factor-1-induced migration toward neural cells, and 20.2% +/- 1.6% displayed firm beta(1)-integrin-mediated adhesion to astrocytes. The latter expressed appropriate adhesion ligands, stabilized beta(1)-integrin expression, and increased beta(2)-integrin expression of HPC. Neural differentiation of HPC could not be identified but astrocytes were able to induce limited self-renewing cell divisions of HPC and thus maintain 25.8% +/- 3.4% of the initial LTC-IC and 80.7% +/- 1.9% of the initial CFC. CONCLUSION: Human HPC are able to interact with neural cells and interaction maintains, albeit to a limited extent, the self-renewal capability of HPC.

Adhesion of hematopoietic progenitor cells to human mesenchymal stem cells as a model for cell-cell interaction.

OBJECTIVE: The significant role of direct contact between hematopoietic progenitor cells (HPC) and the cellular microenvironment for maintaining "stemness" has been demonstrated. Human mesenchymal stem cell (MSC) feeder layers represent a surrogate model for this interaction. Specific adhesion molecules are responsible for this cell-cell contact. METHODS: To define cell-cell contact between HPC and MSC, we have studied adhesive interaction of various fractions of HPC by using a novel assay based on gravitational force upon inversion. Adherent and nonadherent cells were separated and further analyzed with regard to gene expression and long-term hematopoietic culture initiating cell (LTC-IC) frequency. RESULTS: HPC subsets with higher self-renewing capacity demonstrated significantly higher adherence to human MSC (CD34(+) vs CD34(-), CD34(+)/CD38(-) vs CD34(+)/CD38(+), slow dividing fraction vs fast dividing fraction). LTC-IC frequency was significantly higher in the adherent fraction than in the nonadherent fraction. Furthermore, genes coding for adhesion proteins and extracellular matrix were higher expressed in the adherent subsets of CD34(+) cells (fibronectin 1, cadherin 11, vascular cell adhesion molecule-1, connexin 43, integrin beta-like 1, and TGFBI). CONCLUSION: In this study we have demonstrated that primitive subsets of HPC have higher affinity to human MSC. The essential role of specific junction proteins for stabilization of cell-cell contact is indicated by their significant higher expression.

Dynamic cellular phynotyping defines specific mobilization mechanisms of human hematopoietic stem and progenitor cells induced by SDF1α versus synthetic agents.

Efficient mobilization of hematopoietic stem and progenitor cells (HSPC) is one of the most crucial issues for harvesting an adequate amount of peripheral HSPC for successful clinical transplantation. Applying well-defined surrogate models for the bone marrow niche, live cell imaging techniques, and novel tools in statistical physics, we have quantified the functionality of two mobilization agents that have been applied in the clinic, NOX-A12 and AMD3100 (plerixafor), as compared to a naturally occurring chemokine in the bone marrow, SDF1α. We found that NOX-A12, an L-enantiomeric RNA oligonucleotide to SDF1, significantly reduced the adhesion of HSPC to the niche surface mediated via the CXCR4-SDF1α axis, and stretched the migration trajectories of the HSPC. We found that the stretching of trajectories by NOX-A12 was more prominent than that by SDF1α. In contrast, plerixafor exhibited no detectable interference with adhesion and migration. We also found that the deformation of HSPC induced by SDF1α or plerixafor was also drastically suppressed in the presence of NOX-A12. This novel technology of quantitative assessment of "dynamic phenotypes" by physical tools has therefore enabled us to define different mechanisms of function for various extrinsic factors compared to naturally occurring chemokines.

Author Correction: Dynamic cellular phenotyping defines specific mobilization mechanisms of human hematopoietic stem and progenitor cells induced by SDF1α versus synthetic agents.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.