Functional and differential proteomic analyses to identify platelet derived factors affecting ex vivo expansion of mesenchymal stromal cells.

BACKGROUND: Multilineage differentiation, immunomodulation and secretion of trophic factors render mesenchymal stromal cells (MSC) highly attractive for clinical application. Human platelet derivatives such as pooled human platelet lysate (pHPL) and thrombin-activated platelet releasate in plasma (tPRP) have been introduced as alternatives to fetal bovine serum (FBS) to achieve GMP-compliance. However, whereas both pHPL and tPRP support similar proliferation kinetics of lipoaspirate-derived MSC (LA-MSC), only pHPL significantly accelerates bone marrow-derived MSC (BM-MSC) expansion. To identify functionally bioactive factors affecting ex vivo MSC expansion, a differential proteomic approach was performed and identified candidate proteins were evaluated within a bioassay. RESULTS: Two dimensional difference gel electrophoresis (2D-DIGE), MALDI-TOF analyses and complementary Western blotting revealed 20 differential protein species. 14 candidate proteins occured at higher concentrations in pHPL compared to tPRP and 6 at higher concentrations in tPRP. The candidate proteins fibrinogen and apolipoprotein A1 differentially affected LA- and BM-MSC proliferation.In a second set of experiments, reference cytokines known to foster proliferation in FBS were tested for their effects in the human supplements. Interestingly although these cytokines promoted proliferation in FBS, they failed to do so when added to the humanized system. CONCLUSIONS: The differential proteomic approach identified novel platelet derived factors differentially acting on human MSC proliferation. Complementary testing of reference cytokines revealed a lack of stimulation in the human supplements compared to FBS. The data describe a new coherent approach to combine proteomic technologies with functional testing to develop novel, humanized, GMP-compliant conditions for MSC expansion.

Proteomic allergen-peptide/protein interaction assay for the identification of human skin sensitizers.

Modification of proteins by skin sensitizers is a pivotal step in T cell mediated allergic contact dermatitis (ACD). In this process small reactive chemicals interact covalently or non-covalently with cellular or extracellular skin self-proteins or self-peptides to become recognized by the human immune system. Aiming to develop a novel non-animal in vitro test system for predicting sensitization potential of small reactive chemicals in human skin the allergen-peptide/protein interaction assay (APIA) has been developed. By applying modern proteomic technologies together with a target peptide containing all amino acids, the assay permits the profiling of all amino acid specific allergen-peptide interactions. Moreover, potentially crucial allergen-specific Cys-modifications are qualitatively monitored by mass spectrometry and confirmed by a dual peptide approach. Assay conditions chosen mimic the distinct human epidermal reactivity compartments of the skin surface (pH 5.5), stratum basale (pH 6.8), and typical physiological conditions (pH 7.4). An extreme as well as a moderate human contact sensitizer produced Cys-specific mass shifts, whereas a skin irritant did not. Our data indicate that MALDI-MS based and skin-related in vitro technology platforms - like the APIA - are promising tools in developing alternative non-animal allergen assays. This will assist in chemical classification and next generation risk assessment strategies, including REACH and experimental immunotoxicology.

Expansion of Mesenchymal Stem/Stromal cells under xenogenic-free culture conditions.

Mesenchymal Stem/Stromal cells (MSCs) are increasingly applied in cell-based regenerative medicine. To yield clinically relevant cell doses, ex vivo expansion of MSCs is required to be compliant with good manufacturing practice (GMP) guidelines. A lack of standardization and harmonization seems to hamper rapid progress in the translational phase. Most protocols still use fetal bovine serum (FBS) to expand MSCs. However, the high lot-to-lot variability, risk of contamination and immunization call for xenogenic-free culture conditions. Chemically defined media are the ultimate achievement in terms of standardization. These media, however, need to maintain all key cellular and therapy-relevant features of MSCs. Because of the numerous constituents of FBS, the development of such chemically defined media with an optimal composition of the few essential factors is only beginning. Meanwhile, various human blood-derived components are under investigation, including human plasma, human serum, human umbilical cord blood serum and human platelet derivatives such as platelet lysate.

A comparative analysis of the adipogenic potential in human mesenchymal stromal cells from cord blood and other sources.

BACKGROUND AIMS: Mesenchymal stromal cells (MSCs) from umbilical cord blood (CB) attract attention by significantly impaired or absent adipogenic differentiation compared with MSCs derived from bone marrow (BM) and adipose tissue (AT). The diverging adipogenic propensity between the developmentally younger CB-MSCs and MSCs of the adult AT and BM resembles the age-dependent process in the BM, where adipose tissue increases with advancing age, accompanied by loss of bone stability. Thus, MSCs appeal as an attractive model to study the adipogenic process with respect to tissue sources and developmental ages. METHODS: We followed the expression of main adipogenic transcription factors, genes and protein markers in CB-, BM- and AT-MSCs under adipogenic induction, after silencing of preadipocyte factor 1 (Pref-1, PREF1) and after incubation with CB-plasma supplemented adipogenic media. RESULTS: An inverse relation in the expression of adipogenesis-associated markers and PREF1 in CB-MSCs suggested an inhibitory role of Pref-1 toward adipogenesis. However, Pref-1 protein was rarely detected in CB-MSCs, and siRNA silencing of Pref-1 failed to induce adipogenic differentiation in CB-MSCs. Thus, the impaired adipogenic differentiation of CB-MSCs in vitro was unrelated to endogenous Pref-1 protein expression. Nevertheless CB-plasma containing Pref-1 protein revealed an anti-adipogenic effect on AT-MSCs. CONCLUSIONS: Because Pref-1 is vastly abundant in CB-plasma and confers anti-adipogenic properties, Pref-1 in association with the ontogenic milieu probably induces long-lasting unresponsiveness toward adipogenic stimuli in CB-MSCs.

Translating research into clinical scale manufacturing of mesenchymal stromal cells.

It sounds simple to obtain sufficient numbers of cells derived from fetal or adult human tissues, isolate and/or expand the stem cells, and then transplant an appropriate number of these cells into the patient at the correct location. However, translating basic research into routine therapies is a complex multistep process which necessitates product regulation. The challenge relates to managing the expected therapeutic benefits with the potential risks and to balance the fast move to clinical trials with time-consuming cautious risk assessment. This paper will focus on the definition of mesenchymal stromal cells (MSCs), and challenges and achievements in the manufacturing process enabling their use in clinical studies. It will allude to different cellular sources, special capacities of MSCs, but also to current regulations, with a special focus on accessory material of human or animal origin, like media supplements. As cellular integrity and purity, formulation and lot release testing of the final product, validation of all procedures, and quality assurance are of utmost necessity, these topics will be addressed.

Altered gene expression in human adipose stem cells cultured with fetal bovine serum compared to human supplements.

Mesenchymal stromal cells (MSCs) are promising candidates for innovative cell therapeutic applications. For clinical scale manufacturing regulatory agencies recommend to replace fetal bovine serum (FBS) commonly used in MSC expansion media as soon as equivalent alternative supplements are available. We already demonstrated that pooled blood group AB human serum (HS) and thrombin-activated platelet releasate plasma (tPRP) support the expansion of multipotent adipose tissue-derived MSCs (ASCs). Slight differences in size, growth pattern and adhesion prompted us to investigate the level of equivalence by compiling the transcriptional profiles of ASCs cultivated in these supplements. A whole genome gene expression analysis was performed and data verified by polymerase chain reaction and protein analyses. Microarray-based screening of 34,039 genes revealed 102 genes differentially expressed in ASCs cultured with FBS compared to HS or tPRP supplements. A significantly higher expression in FBS cultures was found for 90 genes (fold change ≥2). Only 12 of the 102 genes showed a lower expression in FBS compared to HS or tPRP cultures (fold change ≤0.5). Differences between cells cultivated in HS and tPRP were hardly evident. Supporting previous observations of reduced adhesion of cells cultivated in the human alternatives we detected a number of adhesion and extracellular matrix-associated molecules expressed at lower levels in ASCs cultivated with human supplements. Confirmative assays analyzing transcript or protein expression with selected genes supported these results. Likewise a number of mesodermal differentiation-associated genes were higher expressed in cells grown in FBS. Quantifying adipogenic and osteogenic differentiation lacked to demonstrate a clear correlation to the supplement due to donor-specific variances. Our results emphasize the necessity of comparability studies as they indicate that FBS induces a culture adaptation exceeding that of ex vivo culture in human supplements and thus may contribute to the therapeutic potential.