Eliminating the need of serum testing using low serum culture conditions for human bone marrow-derived mesenchymal stromal cell expansion.

BACKGROUND: The conventional expansion of human mesenchymal stromal cells (hMSC) for tissue engineering or (pre-) clinical investigation includes the use of 10% fetal bovine serum (FBS). However, there exists immense lot-to-lot variability in FBS samples and time consuming as well as cost intensive lot pre-testing is essential to guarantee optimal hMSC proliferation and stem cells characteristics maintenance. Furthermore, lot-to-lot variability impedes the long-term consistency of research and comparability between research groups. Therefore, we investigated the use of defined, invariable, non-synthetic FBS in low serum culture conditions for isolation and expansion of hMSC. METHODS: hMSC were isolated from bone marrow in Panserin 401 supplemented with growth factors and 2% MSC-tested or non-tested, defined, invariable, non-synthetic FBS and further cultivated in vitro. The surface marker expression, differentiation capacity as well as cell proliferation and cytotoxicity was analyzed and compared between serum samples. RESULTS: Cells isolated and cultivated with low concentrations of MSC-tested or non-tested FBS demonstrated no differences in surface marker expression or differentiation capacity. Proliferation of hMSC was equal in medium supplemented with either serum with no indication of cell death. CONCLUSIONS: The low serum concentration in Panserin 401 supplemented with growth factors enables the use of defined, invariable, non-synthetic FBS for the isolation and expansion of hMSC. The required hMSC characteristics like surface marker expression and differentiation capacity are maintained. Importantly, no differences in the cell proliferation could be detected. Therefore, using these low-serum culture conditions, the need for lot-to-lot pre-testing of FBS usually needed for optimal hMSC expansion is abolished leading to long-term consistency and comparability of results.

Microstructure and cytocompatibility of collagen matrices for urological tissue engineering.

OBJECTIVES: • To analyse the in vitro cytocompatibility of several engineered collagen-based biomaterials for tissue engineering of the urinary tract. • Tissue-engineered implants for the reconstruction of the urinary tract are of major interest for urological researchers as well as clinicians. Although several materials have been investigated, the ideal replacement has still to be identified. MATERIALS AND METHODS: • Several collagen matrices were tested. • Electron microscopy was used to visualize the microstructure of the tested matrices. • Examination of cell attachment and growth of primary porcine urothelial and smooth muscle cells were performed and cell phenotypes were analysed using immunohistochemical stains. • Urea permeability was investigated using Ussing chamber experiments. RESULTS: • The best cytocompatibility for both urinary tract-specific cell types was obtained with OptiMaix(®) (Matricel GmbH, Herzogenrath, Germany) materials. • Cell-specific phenotypes were maintained during culture as shown by immunohistochemical staining. • Furthermore, simultaneous cultivation of both cell types for 7 and 14 days significantly reduced urea permeability. CONCLUSION: • These results show the potential of OptiMaix materials in tissue engineering approaches of urinary tract tissues.

Expansion of human bone marrow-derived mesenchymal stromal cells: serum-reduced medium is better than conventional medium.

BACKGROUND AIMS: Human mesenchymal stromal cells (hMSC) are of enormous interest for various clinical applications. For the expansion of isolated hMSC to relevant numbers for clinical applications, 10% fetal bovine serum (FBS)-supplemented medium is commonly used. The main critical disadvantage of FBS is the possibility of transmission of infectious agents as well as the possibility of immune rejection of the transplanted cells in response to the bovine serum. Therefore, we tested a commercially available medium, Panserin 401, that was specifically developed for serum-free cell cultivation. METHODS: hMSC were isolated from bone marrow (BM) and expanded in either Dulbecco's modified Eagle medium (DMEM) or Panserin 401 alone, or combined with FBS (2% or 10%), with or without supplementary growth factors. Cell proliferation and cytotoxicity were monitored twice a week for 3 weeks. RESULTS AND CONCLUSIONS: No proliferation was observed in any of the serum-free media. However, DMEM/10% FBS (the conventional culture medium for hMSC) and DMEM/2% FBS with growth factors revealed moderate proliferation. Interestingly, the best proliferation was obtained using Panserin 401 supplemented with 2% FBS and growth factors (as well as with 10% FBS). Analysis of cell growth in Panserin 401 supplemented with 2% FBS only or with growth factors only revealed no proliferation, demonstrating the necessity of the combination of 2% FBS and growth factors. Efficient isolation and expansion of hMSC from cancellous bone could also be performed using Panserin 401 with 2% FBS and growth factors. Furthermore, these isolated cultures maintained multipotency, as demonstrated by adipogenic and osteogenic differentiation.

Growth factor and cytokine expression of human mesenchymal stromal cells is not altered in an in vitro model of tissue damage.

BACKGROUND AIMS: The beneficial effect of human (h) mesenchymal stromal cell (MSC) transplantation in a variety of cell-based intervention strategies is widely believed to be because of paracrine mechanisms. The modification of hMSC cytokine and growth-factor expression patterns were studied following exposure to lipopolysaccharide (LPS) and tissue homogenates (representing tissue debris) from normal and pathologic tissues. METHODS: Human bone marrow-derived MSC were stimulated with LPS or exposed to homogenate from normal or pathologic rat spinal cord or heart. The expression profiles of a number of cytokines and growth factors were investigated using quantitative reverse transcription (RT)-polymerase chain reaction (PCR) with human-specific primers. The effects of tissue homogenates on hMSC proliferation and migratory behavior were also investigated. RESULTS: Stimulation of hMSC with LPS resulted in an up-regulation of interleukin (IL)-1ß, IL-6 and IL-8. However, the pattern of up-regulation varied between donor samples. Furthermore, LPS treatment resulted in a donor-dependent alteration of growth factor expression. Induction of a shift in expression pattern was not observed following exposure to homogenates from either normal or pathologic tissues. Tissue homogenates did stimulate cell proliferation, but not migration. CONCLUSIONS: The hMSC expression pattern is apparently stable, even when cells are confronted by debris from different tissue types. However, treatment of hMSC with LPS is able to change the expression of cytokines and growth factors in a donor-dependent manner that may enhance their potential use in regenerative medicine.

Application of mesenchymal stromal cells in urological diseases.

The application of stem cells and their use in tissue-engineering approaches is emerging in clinical therapeutic intervention strategies. The use of adult stem cells, either autologous or allogenic, does not raise ethical concerns, in contrast to embryonic stem cells. Mesenchymal stromal cells (MSCs) can be easily obtained from bone marrow or from adipose tissue and further expanded in vitro. Due to their differentiation capacity, MSCs are very attractive for tissue engineering purposes. Furthermore, MSCs secrete a variety of mediators that have beneficial effects on the regenerating tissue. In this review we give an insight into stem cell hierarchy, define the properties of MSCs and summarize recent reports of their administration in urological diseases.

Neural differentiation potential of human bone marrow-derived mesenchymal stromal cells: misleading marker gene expression.

BACKGROUND: In contrast to pluripotent embryonic stem cells, adult stem cells have been considered to be multipotent, being somewhat more restricted in their differentiation capacity and only giving rise to cell types related to their tissue of origin. Several studies, however, have reported that bone marrow-derived mesenchymal stromal cells (MSCs) are capable of transdifferentiating to neural cell types, effectively crossing normal lineage restriction boundaries. Such reports have been based on the detection of neural-related proteins by the differentiated MSCs. In order to assess the potential of human adult MSCs to undergo true differentiation to a neural lineage and to determine the degree of homogeneity between donor samples, we have used RT-PCR and immunocytochemistry to investigate the basal expression of a range of neural related mRNAs and proteins in populations of non-differentiated MSCs obtained from 4 donors. RESULTS: The expression analysis revealed that several of the commonly used marker genes from other studies like nestin, Enolase2 and microtubule associated protein 1b (MAP1b) are already expressed by undifferentiated human MSCs. Furthermore, mRNA for some of the neural-related transcription factors, e.g. Engrailed-1 and Nurr1 were also strongly expressed. However, several other neural-related mRNAs (e.g. DRD2, enolase2, NFL and MBP) could be identified, but not in all donor samples. Similarly, synaptic vesicle-related mRNA, STX1A could only be detected in 2 of the 4 undifferentiated donor hMSC samples. More significantly, each donor sample revealed a unique expression pattern, demonstrating a significant variation of marker expression. CONCLUSION: The present study highlights the existence of an inter-donor variability of expression of neural-related markers in human MSC samples that has not previously been described. This donor-related heterogeneity might influence the reproducibility of transdifferentiation protocols as well as contributing to the ongoing controversy about differentiation capacities of MSCs. Therefore, further studies need to consider the differences between donor samples prior to any treatment as well as the possibility of harvesting donor cells that may be inappropriate for transplantation strategies.

Cell-cell interactions of human neural progenitor-derived astrocytes within a microstructured 3D-scaffold.

In the present in vitro study, the axon growth promoting effects of human neural progenitor-derived astrocytes (hNP-AC) were investigated in simple 2D- as well as in more complex 3D-culture systems. The interactions of the hNP-AC with migrating Schwann cells and fibroblasts were also studied. hNP-AC were found to promote extensive dorsal root ganglion axon regeneration in 2D cultures, being even greater than that observed on the positive control, laminin-coated substrate. Contact-mediated mechanisms and the release of substances into the medium both played a role in supporting axon regeneration. Following seeding onto 3D collagen scaffolds, hNP-AC also promoted significantly greater axon regeneration from DRG explants than was seen on non-seeded scaffolds. The highly orientated, porous microstructure of the scaffold also supported substantial intermixing of hNP-AC and migrating Schwann cells/fibroblasts from the DRG explant, cell populations that are normally mutually repulsive. This suggests that the topography of 3D scaffolds may not only influence cell-substrate interactions but also cell-cell interactions within the scaffold. This opens the possibility that the design of future scaffolds could be optimised to enhance cell integration as well as modulating complex cell-cell interactions.

Axon growth-promoting properties of human bone marrow mesenchymal stromal cells.

Mesenchymal stromal cells are promising candidate donor cells for promoting functional tissue repair following traumatic spinal cord injury (SCI), however, the mechanism(s) of action remain poorly defined. Here, we describe an in vitro study of the axon growth-promoting properties of highly enriched populations of adult human mesenchymal stromal cells (hMSC). A random, non-oriented pattern of neuritic outgrowth was observed from dissociated adult rat DRG neurons seeded onto confluent A431 cells and PLL/laminin positive control substrata. Confluent hMSC formed arrays of similarly orientated cell bodies and processes which supported the regeneration of significantly more primary neurites but a slightly lower overall neuritic length than was observed over the PLL/laminin control substrate. The hMSC exerted a strong influence on the direction of neuritic outgrowth, with many regenerating processes following the orientation of underlying hMSC. The production of extracellular matrix appeared to be responsible for neuritic directionality, but the release of growth factors was a significant promoter for DRG neuritic outgrowth. This suggests that further investigations into the properties of hMSC may be of particular interest in the development of transplant-mediated strategies intending to promote functional axonal regeneration after SCI.