Reconstitution of bone-like matrix in osteogenically differentiated mesenchymal stem cells-collagen constructs: a three-dimensional in vitro model to study hematopoietic stem cell niche

published_or_final_versio

Bi-directional cell-pericellular matrix interactions direct stem cell fate

Modifiable hydrogels have revealed tremendous insight into how physical characteristics of cells’ 3D environment drive stem cell lineage specification. However, in native tissues, cells do not passively receive signals from their niche. Instead they actively probe and modify their pericellular space to suit their needs, yet the dynamics of cells’ reciprocal interactions with their pericellular environment when encapsulated within hydrogels remains relatively unexplored. Here, we show that human bone marrow stromal cells (hMSC) encapsulated within hyaluronic acid-based hydrogels modify their surroundings by synthesizing, secreting and arranging proteins pericellularly or by degrading the hydrogel. hMSC’s interactions with this local environment have a role in regulating hMSC fate, with a secreted proteinaceous pericellular matrix associated with adipogenesis, and degradation with osteogenesis. Our observations suggest that hMSC participate in a bi-directional interplay between the properties of their 3D milieu and their own secreted pericellular matrix, and that this combination of interactions drives fate

The dominant Anopheles vectors of human malaria in the Asia-Pacific region: occurrence data, distribution maps and bionomic précis

<p>Abstract</p> <p>Background</p> <p>The final article in a series of three publications examining the global distribution of 41 dominant vector species (DVS) of malaria is presented here. The first publication examined the DVS from the Americas, with the second covering those species present in Africa, Europe and the Middle East. Here we discuss the 19 DVS of the Asian-Pacific region. This region experiences a high diversity of vector species, many occurring sympatrically, which, combined with the occurrence of a high number of species complexes and suspected species complexes, and behavioural plasticity of many of these major vectors, adds a level of entomological complexity not comparable elsewhere globally. To try and untangle the intricacy of the vectors of this region and to increase the effectiveness of vector control interventions, an understanding of the contemporary distribution of each species, combined with a synthesis of the current knowledge of their behaviour and ecology is needed.</p> <p>Results</p> <p>Expert opinion (EO) range maps, created with the most up-to-date expert knowledge of each DVS distribution, were combined with a contemporary database of occurrence data and a suite of open access, environmental and climatic variables. Using the Boosted Regression Tree (BRT) modelling method, distribution maps of each DVS were produced. The occurrence data were abstracted from the formal, published literature, plus other relevant sources, resulting in the collation of DVS occurrence at 10116 locations across 31 countries, of which 8853 were successfully geo-referenced and 7430 were resolved to spatial areas that could be included in the BRT model. A detailed summary of the information on the bionomics of each species and species complex is also presented.</p> <p>Conclusions</p> <p>This article concludes a project aimed to establish the contemporary global distribution of the DVS of malaria. The three articles produced are intended as a detailed reference for scientists continuing research into the aspects of taxonomy, biology and ecology relevant to species-specific vector control. This research is particularly relevant to help unravel the complicated taxonomic status, ecology and epidemiology of the vectors of the Asia-Pacific region. All the occurrence data, predictive maps and EO-shape files generated during the production of these publications will be made available in the public domain. We hope that this will encourage data sharing to improve future iterations of the distribution maps.</p

Recapitulation of mesenchymal cell condensation using human mesenchymal stem cell-collagen microspheres.

Congress Theme: Past, Present, Future: the evolution of Regenerative MedicinePoster abstractsMesenchymal cell condensation is a critical transitional stage that precedes cartilage or bone formation. A microencapsulation technique was previously established to entrap mesenchymal stem cells (MSC) in collagen microspheres. Here, we hypothesize that the MSC-induced gel contraction mimics the mesenchymal cell condensation and thereby regulates MSC differentiation. The effect of cell density and timing for chondrogenesis induction was investigated. In general, gene expression of SOX9 was upregulated at early time points (day 0–2) while RUNX2 was gradually increased. COL2A1 was constantly expressed in intermediate cell density and was increased at later time points in high cell density but decreased in low cell density. COL1A1 was downregulated except the intermediate cell density with constant expression. COL10A1 was constantly expressed except the low cell density with a decreasing trend. Osterix was gradually upregulated and peak at day 7. However, aggrecan and ALP expression was lower than the monolayer throughout the culturing period. Nuclear localization of Sox9 was observed at day 0 and day 1 whereas nuclear localization of Runx2 was observed from day 0 to day 7 in low and intermediate cell densities but only at day 0 in high cell density. Deposition of collagen II and collagen X were increased with time and cell density. Upon chondrogenesis, gene expression of SOX9, COL2A1, and aggrecan were higher in MSC-collagen microspheres when chondrogenesis was induced at day 3 than that at day 0 and day 1. This work suggests the importance of timing of induction in developing protocols for stem cell chondrogenesis

Cellular stress response of intervertebral cells under mechanical loading

Poster Presenatio

Loading-Induced Stress Response in the Intervertebral Disc

Conference theme: The Intervertebral Disc - from Degeneration to Therapeutic Motion PreservationThe abstract can be viewed at http://www.spineresearchforum.org/WFSR_2014_Thieme_AbstractBook_with_Cover.pdfIntroduction Previous research has been done to study the effect of mechanical loading on intervertebral disc (IVD) cells. However, few studies have investigated in whether the IVD cells perceive mechanical loading as stress and respond by expression of stress response proteins such as heat shock proteins (HSP). Studies have shown that stress response can be seen in cell line chondrocytes under hydrostatic pressure. On the other hand, studies have also shown that expression of heat shock protein-72 (HSP72) and HSP27was associated with disc degeneration and IVD cells can secrete HSP70 in response to oxidative stress. This study aims to study the stress response in the IVD in response to compressive loading and whether the disc cells are able to adapt to the loading. The outcome of the study will help to understand how the disc cells adapt or cope with mechanical stress. Materials and Methods Fresh adult bovine caudal discs were harvested and cultured with dynamic compressive loading applied at physiological range magnitude, 0.1 to 0.6 MPa. The culture condition was such that the discs underwent 2 hours of dynamic loading, followed by 22 hours of resting for 2 days. Samples were retrieved at different time points: right after loading (Dyna) and right after resting (DyNa+rest). Positive control discs were put under static loading (0.35 MPa, static) and heat shock (43° C, HS) exposed for 2 h/d during 2 days and gene expression was quantified right after the treatments. Both nucleus pulposus (NP) and annulus fibrosus (AF) were retrieved for gene expression study of the cellular stress response genes. HSP72 and heat shock factor-1 (HSF1). HSP72 is the general stress response protein which is upregulated in the cell in response to stress while HSF1 is the transcriptional factor of HSP72. The expression was normalized to free swelling control. Results In the NP of the bovine disc, both positive controls (HS and static) expressed high level of HSP72, confirming their expression in the NP tissues and their response to stress. For the experimental groups, the expression of HSP72 was upregulated after loading, decreased after resting but was again increased after second round of loading at day 2. On the other hand, HSF1 expression increased after resting in the day 1 loading and peaked at day 2 after loading. For the AF tissues, the expression of HSF1 was low in most of the groups including the positive control, even the HSP72 expression was high in these two groups. The expression of HSP72 in AF tissues was decreasing with both resting and an additional round of loading. The pattern of HSF1 expression of AF tissues was similar to the NP tissues where the expression was the highest 2 days after loading. Conclusion This study showed that the IVD cells do upregulate the stress response proteins expression in response to loading induced stress. The cells express HSP72 in response to the stress while HSF1 may have a slower and transient expression. The increase in HSP72 and HSF1 expression after two rounds of loading may indicate more cycles are needed to see whether there is adaptation in stress response induced by mechanical loading. Disclosure of Interest None declare

Development of protein-based 3D culture system for bovine nucleus pulposus cells

Congress Theme: The Evolution of Regenerative Medicin

Development of three-dimensional culture system for bovine nucleus pulposus cells

Poster abstractsCongress Theme: Past, Present, Future: the evolution of Regenerative MedicineINTRODUCTION: Nucleus pulposus is a central component of the intervertebral disc and rich in extracellular matrix (ECM). In the pathology of degenerative disc disease (DDD), the changes in ECM may be related to changes in nucleus pulposus cells (NPCs). To study NPCs, it is important to culture them in vitro in an environment similar to the native tissue. Many factors such as the interactions between NPCs and ECM, the topological cues and the mechanical properties of the constructs should be considered. If a 3D NPCmatrix system mimicking the native NP is developed, it can be used in future studies in regenerative medicine and DDD as a model platform. OBJECTIVE: The objective is to maintain the survival and phenotype of NPCs in a 3D culture system, by resembling the native tissue microenvironment. Specifically, we compare two starting materials, namely collagen and collagen-GAG constructs. METHODOLOGY: bNPCs were either encapsulated in collagen microspheres or seeded on collagen-GAG constructs fabricated by coprecipitation. They were cultured up to two weeks before evaluation. Cell morphology, F-actin distribution and viability were revealed by staining. Phenotype and components in the ECM were analyzed by qRT-PCR, immunohistochemistry and histology. RESULTS: bNPCs can survive, proliferate and maintain some phenotypic markers, in our 3D systems. Collagen-GAG constructs may be better than collagen alone for long term culture of bNPCs. CONCLUSION: Collagen-GAG constructs may be a good starting material for culturing NPCs. Including other ECM components into the culture system may further help in supporting the cell phenotype

Three-dimensional culture of bovine nucleus pulposus cells in protein matrices

Poster PresentationConference Theme: The Extracellular Matrix NicheIntroduction and objectives Nucleus pulposus (NP) is located at the center of the intervertebral disc (IVD) and rich in collagen type II and proteoglycans. The glycosaminoglycans (GAGs) in the proteoglycans enable the retention of a high amount of water, which is important for the mechanical function of the IVD. In the pathology of degenerative disc disease (DDD), the loss of proteoglycan and water may be related to changes in NP cells (NPCs). If a three-dimensional NPC-matrix platform mimicking the native NP is developed, it can be used in future research in tissue engineering and serve as a model platform to study NPCs in DDD. Methods NP from bovine caudal spines was digested to isolate bovine NPCs (bNPCs). The bNPCs were encapsulated in microspheres made of collagen type I and then cultured and fixed on days 3, 7, 10 and 14. Immunohistochemistry of collagen type II and histology including Alcian blue and hematoxylin and eosin (H&E) staining were done. Immunohistochemistry of NPC markers will also be done. The viability of the bNPCs in the microspheres was evaluated by live/dead staining. Besides the microsphere system, another three-dimensional culture system based on collagen-GAG co-precipitate will be investigated. In brief, chondroitin-6-sulfate, aminated type I collagen and bNPCs will be mixed together followed by centrifugation to collect the constructs, which will be cultured and evaluated in the same ways as the microspheres. Results H&E showed that in the microspheres, the bNPCs were sparse in day 3 and became dense starting from day 7. At the periphery of the microspheres, a ring with very dense cells was formed since day 7 and grew thicker in later time points. The cell density in the ring was higher than that in the central region. Any difference between the cells in the two regions is yet to be investigated. Most of the bNPCs became highly elongated since day 10. Alcian blue staining was very weak and weak on days 3 and 7, respectively. It became moderate on days 10 and 14. These indicated that GAGs were slowly accumulated in the microspheres. Very intense staining for collagen type II was observed on days 7, 10 and 14. Live/dead staining showed that most of the cells were alive in all time points. Conclusions The collagen microspheres supported the survival and proliferation of bNPCs and maintained some of the phenotypic characteristics of bNPCs, including the production of collagen type II and GAGs