an-enzymatic-method-to-rescue-mesenchymal-stem-cells-from-clotted

An Enzymatic Method to Rescue Mesenchymal Stem Cells from Clotted Bone Marrow Samples

Mesenchymal stem cells (MSCs) - usually obtained from bone marrow - often require expansion culture. Our protocol uses clinical grade urokinase to degrade ...

Swiss Paraplegic Research

The capacity of mesenchymal stem cells (MSCs) to differentiate into intervertebral disc (IVD)-like cells has been well described, but their ability to modulate the inflammatory processes in the IVD remains unclear. We found that tissue obtained by discectomy of degenerated and post-traumatic IVD contains significant amounts of IgG antibodies, a sign of lymphocyte infiltration. Further we investigated whether MSCs in vitro, which were characterized for their multilineage differentiation potential and may have immunomodulatory effects on IVD fragments. IVD fragments were co-cultured in contact with peripheral blood lymphocytes (PBLs) and MSCs, and as functional controls we used contact co-cultures of PBLs stimulated with pokeweed mitogen (2.5 μg/mL) and MSCs. The time course of lymphocyte proliferation (Alamar Blue), IgG (ELISA) and gene expression (RT-PCR) of anti-inflammatory cytokines (TGF-β1, IL-10) by MSCs and pro-inflammatory molecules (IL-1α, IL-1β and TNF-α) by the IVD fragments were analyzed. Depending on the response to the presence of MSCs, the IVD fragments (n = 13) were divided in two groups: responders (n = 9), where inflammation was inhibited by MSCs and non-responders (n = 4), where MSCs did not decrease inflammation. At 1 week in co-culture, MSCs reduced significantly the IgG production in the IVD responders group to 69% and PBLs proliferation to 57% of the control. MSCs expression of the anti-inflammatory TGF-β1 increased with time, while IL-10 was expressed only at day 1. IVD gene expression of TNF-α decreased constantly, whereas IL-1α and IL-1β expression increased. In conclusion, these data suggest that MSCs may modulate disc-specific inflammatory and pain status and aid regeneration of the host tissue.

Human mesenchymal stem cell co-culture modulates the immunological properties of human intervertebral disc tissue fragments in vitro.

Degeneration of intervertebral discs (IVD) is one of the main causes of back pain and tissue engineering has been proposed as a treatment. Tissue engineering requires the use of highly expensive growth factors, which might, in addition, lack regulatory approval for human use. In an effort to find readily available differentiation factors, we tested three molecules--dexamethasone, triiodothyronine (T3) and insulin--on human IVD cells isolated after surgery, expanded in vitro and transferred into alginate beads. Triplicates containing 40 ng/ml dexamethasone, 10 nM T3 and 10 µg/ml insulin, together with a positive control (10 ng/mL transforming growth factor (TGF)-beta 1), were sampled weekly over six weeks and compared to a negative control. Furthermore, we compared the results to cultures with optimized chondrogenic media and under hypoxic condition (2% O2). Glycosaminoglycan (GAG) determination by Alcian Blue assay and histological staining showed dexamethasone to be more effective than T3 and insulin, but less than TGF-beta1. DNA quantification showed that only dexamethasone stimulated cell proliferation. qPCR demonstrated that TGF-beta1 and the optimized chondrogenic groups increased the expression of collagen type II, while aggrecan was stimulated in cultures containing dexamethasone. Hypoxia increased GAG accumulation, collagen type II and aggrecan expression, but had no effect on or even lowered cell number. In conclusion, dexamethasone is a valuable and cost-effective molecule for chondrogenic and viability induction of IVD cells under normoxic and hypoxic conditions, while insulin and T3 did not show significant differences.

The in vitro effects of dexamethasone, insulin and triiodothyronine on degenerative human intervertebral disc cells under normoxic and hypoxic conditions.

Cell-based therapies for regeneration of the degenerated intervertebral disc (IVD) are an alternative to current surgical intervention. Mesenchymal stem cells (MSCs), in combination with a scaffold, might be ideal candidates for regenerating nucleus pulposus (NP), the pressure-distributing part of the IVD. While the use of growth factors for MSCs differentiation currently receives major attention, in this study we compare the performance of sponge-like matrixes in supporting cell differentiation into NP-like cells.

Influence of different commercial scaffolds on the in vitro differentiation of human mesenchymal stem cells to nucleus pulposus-like cells.

Protein synthesis is a tightly regulated, energy-consuming process. The control of mRNA translation into protein is fundamentally important for the fine-tuning of gene expression; additionally, precise translational control plays a critical role in many cellular processes, including development, cellular growth, proliferation, differentiation, synaptic plasticity, memory, and learning. Eukaryotic translation initiation factor 4h (Eif4h) encodes a protein involved in the process of protein synthesis, at the level of initiation phase. Its human homolog, WBSCR1, maps on 7q11.23, inside the 1.6 Mb region that is commonly deleted in patients affected by the Williams-Beuren syndrome, which is a complex neurodevelopmental disorder characterized by cardiovascular defects, cerebral dysplasias and a peculiar cognitive-behavioral profile. In this study, we generated knockout mice deficient in Eif4h. These mice displayed growth retardation with a significant reduction of body weight that began from the first week of postnatal development. Neuroanatomical profiling results generated by magnetic resonance imaging analysis revealed a smaller brain volume in null mice compared with controls as well as altered brain morphology, where anterior and posterior brain regions were differentially affected. The inactivation of Eif4h also led to a reduction in both the number and complexity of neurons. Behavioral studies revealed severe impairments of fear-related associative learning and memory formation. These alterations suggest that Eif4h might contribute to certain deficits associated with Williams-Beuren syndrome.

Growth defects and impaired cognitive-behavioral abilities in mice with knockout for Eif4h, a gene located in the mouse homolog of the Williams-Beuren syndrome critical region.

Human bone marrow-derived mesenchymal stem cells (MSCs) have limited growth potential in vitro and cease to divide due to replicative senescence, which from a tissue-engineering perspective has practical implications, such as defining the correct starting points for differentiation and transplantation. Time spent in culture before the loss of required differentiation potential is different and reflects patient variability, which is a problem for cell expansion. This study aimed to develop a score set which can be used to quantify the senescent state of MSCs and predict whether cells preserve their ability to differentiate to osteogenic, adipogenic and chondrogenic phenotypes, based on colony-forming unit (CFU) assay, population doubling time (PDT), senescence-associated β-galactosidase (SA-β-Gal) activity, cell size, telomere length and gene expression of MSCs cultured in vitro over 11 passages. This set of morphological, physiological and genetic senescence markers was correlated to the ability of MSCs to differentiate. Differentiation efficiency was assessed by marker genes and protein expression. CFUs decreased with increasing passage number, whereas SA-β-Gal activity and PDT increased; however, the correlation with MSCs' differentiation potential was sometimes unexpected. The expression of genes related to senescence was higher in late-passage cells than in early-passage cells. Early-passage cells underwent efficient osteogenic differentiation, with mid-passage cells performing best in chondrogenic differentiation. Late-passage cells preserve only adipogenic differentiation potential. Based on this marker set, we propose a senescence score in which combined markers give a reliable quality control of MSCs, not depending only on mechanistic passage number. Copyright © 2013 John Wiley & Sons, Ltd.

An in vitro expansion score for tissue-engineering applications with human bone marrow-derived mesenchymal stem cells.

Testosterone (T) is a hormone and regulator involved in the processes of development of the organism (ie, promoting development of bone and muscle mass). Although T effects on the mesenchyme-derived muscle, bone, and adipose tissues are well studied, T effects on intervertebral disc (IVD) have not been reported.

Physiological testosterone levels enhance chondrogenic extracellular matrix synthesis by male intervertebral disc cells in vitro, but not by mesenchymal stem cells.

The study of canine bone marrow-derived mesenchymal stem cells (MSC) has a prominent position in veterinary cell-based applications. Yet the plethora of breeds, their different life spans and inter-breed variations provide unclearness on what can be achieved specifically by such therapies. In this study, we compared a set of morphological, physiological, and genetic markers of MSC derived from large dog breeds, namely: Border collie, German shepherd, Labrador, Malinois, Golden retriever and Hovawart. We compared colony forming units (CFU) assay, population doubling time (PDT), senescence-associated β-galactosidase (SA-β-Gal) activity, telomere length and gene expression of MSC, as well as the ability of cells to differentiate to osteogenic, adipogenic and chondrogenic phenotypes. The influence of the culture media α-MEM, low glucose DMEM and high glucose DMEM, used in cell isolation and expansion, was investigated in presence and absence of basic Fibroblast Growth Factor (bFGF). Initial cell yield was not affected by culturing medium, but MSC expanded best in α-MEM supplemented with bFGF. After isolation, the number of MSC was similar among breeds - as shown by equivalent CFU - except in the Hovawart samples, which had fivefold less CFU. Telomere lengths was similar among breeds. MSC divided actively only for four weeks in culture (PDT = ~50 hours/division), except Border collie cells divided for longer time than cells from other groups. The percentage of senescent cells increased linearly in all breeds with time, with a faster rate in German shepherd, Labrador and Golden retriever. Border collie cells underwent efficient osteogenic differentiation, Hovawart cells performed the best in chondrogenic differentiation and Labrador cells in both, while German shepherd cells had the lower differentiation potential. MSC from all breed preserved the same adipogenic differentiation potential. In conclusion, despite variations, isolated MSC can be expanded and differentiated in vitro and all breeds are eligible for MSC-based therapies.

Canine Mesenchymal Stem Cell Potential and the Importance of Dog Breed - Implication for Cell-based Therapies.

Mesenchymal stem cells (MSCs) are expected to have a fundamental role in future cell-based therapies because of their high proliferative ability, multilineage potential, and immunomodulatory properties. Autologous transplantations have the "elephant in the room" problem of wide donor variability, reflected by variability in MSC quality and characteristics, leading to uncertain outcomes in the use of these cells. We propose life imaging as a tool to characterize populations of human MSCs. Bone marrow MSCs from various donors and in vitro passages were evaluated for their in vitro motility, and the distances were correlated to the adipogenic, chondrogenic, and osteogenic differentiation potentials and the levels of senescence and cell size. Using life-image measuring of track lengths of 70 cells per population for a period of 24 hours, we observed that slow-moving cells had the higher proportion of senescent cells compared with fast ones. Larger cells moved less than smaller ones, and spindle-shaped cells had an average speed. Both fast cells and slow cells were characterized by a low differentiation potential, and average-moving cells were more effective in undergoing all three lineage differentiations. Furthermore, heterogeneity in single cell motility within a population correlated with the average-moving cells, and fast- and slow-moving cells tended toward homogeneity (i.e., a monotonous moving pattern). In conclusion, in vitro cell motility might be a useful tool to quickly characterize and distinguish the MSC population's differentiation potential before additional use.

Alessandro Bertolo

Swiss Paraplegic Research

An Enzymatic Method to Rescue Mesenchymal Stem Cells from Clotted Bone Marrow Samples