Pedro Z. Andrade

Ex vivo expansion of human mesenchymal stem cells: A more effective cell proliferation kinetics and metabolism under hypoxia

The low bone marrow (BM) MSC titers demand a fast ex vivo expansion process to meet the clinically relevant cell dosage. Attending to the low oxygen tension of BM in vivo, we studied the influence of hypoxia on human BM MSC proliferation kinetics and metabolism. Human BM MSC cultured under 2% (hypoxia) and 20% O2 (normoxia) were characterized in terms of proliferation, cell division kinetics and metabolic patterns. BM MSC cultures under hypoxia displayed an early start of the exponential growth phase, and cell numbers obtained at each time point throughout culture were consistently higher under low O2, resulting in a higher fold increase after 12 days under hypoxia (40 ± 10 vs. 30 ± 6). Cell labeling with PKH26 allowed us to determine that after 2 days of culture, a significant higher cell number was already actively dividing under 2% compared to 20% O2 and BM MSC expanded under low oxygen tension displayed consistently higher percentages of cells in the latest generations (generations 4–6) until the 5th day of culture. Cells under low O2 presented higher specific consumption of nutrients, especially early in culture, but with lower specific production of inhibitory metabolites. Moreover, 2% O2 favored CFU‐F expansion, while maintaining BM MSC characteristic immunophenotype and differentiative potential. Our results demonstrated a more efficient BM MSC expansion at 2% O2, compared to normoxic conditions, associated to an earlier start of cellular division and supported by an increase in cellular metabolism efficiency towards the maximization of cell yield for application in clinical settings. J. Cell. Physiol. 223: 27–35, 2010.

Toward a Clinical-Grade Expansion of Mesenchymal Stem Cells from Human Sources: A Microcarrier-Based Culture System Under Xeno-Free Conditions

The immunomodulatory properties of mesenchymal stem cells (MSCs) make them attractive therapeutic agents for a wide range of diseases. However, the highly demanding cell doses used in MSC clinical trials (up to millions of cells/kg patient) currently require labor intensive methods and incur high reagent costs. Moreover, the use of xenogenic (xeno) serum-containing media represents a risk of contamination and raises safety concerns. Bioreactor systems in combination with novel xeno-free medium formulations represent a viable alternative to reproducibly achieve a safe and reliable MSC doses relevant for cell therapy. The main goal of the present study was to develop a complete xeno-free microcarrier-based culture system for the efficient expansion of human MSC from two different sources, human bone marrow (BM), and adipose tissue. After 14 days of culture in spinner flasks, BM MSC reached a maximum cell density of (2.0±0.2)×10⁵ cells·mL⁻¹ (18±1-fold increase), whereas adipose tissue-derived stem cells expanded to (1.4±0.5)×10⁵ cells·mL⁻¹ (14±7-fold increase). After the expansion, MSC expressed the characteristic markers CD73, CD90, and CD105, whereas negative for CD80 and human leukocyte antigen (HLA)-DR. Expanded cells maintained the ability to differentiate robustly into osteoblast, adipocyte, and chondroblast lineages upon directed differentiation. These results demonstrated the feasibility of expanding human MSC in a scalable microcarrier-based stirred culture system under xeno-free conditions and represent an important step forward for the implementation of a Good Manufacturing Practices-compliant large-scale production system of MSC for cellular therapy.

Maximizing the ex vivo expansion of human mesenchymal stem cells using a microcarrier-based stirred culture system.

Bioreactor systems have been developed as alternatives to standard culture flasks due to their homogeneous nature, easiness of monitoring and increased cell production. Here we investigated the in vitro expansion of bone marrow (BM) mesenchymal stem cells (MSC) in spinner flasks, using gelatin microcarriers (Cultispher S) to support cell adhesion and proliferation. MSC expansion was performed using a low-serum containing medium (2% of fetal bovine serum, FBS). A strategy was defined for the maximization of cell expansion: microcarriers were pre-coated with FBS in order to increase cell seeding efficiency and an adequate feeding regime was established (25% medium exchange everyday). The maximum cell density, 4.2 x 10(5)cells/mL, was obtained at day 8, corresponding to a fold increase in total cell number of 8.4+/-0.8. Expanded MSC retained their differentiation potential into adipogenic and osteogenic lineages, as well as their clonogenic ability. Harvested cells expressed >90% of CD73, CD90 and CD105 markers. These results demonstrated that a microcarrier-based stirred culture system is adequate for human MSC expansion, using a low-serum containing medium, allowing the generation of significant cell numbers for potential applications in regenerative medicine.

Mesenchymal stem cells from umbilical cord matrix, adipose tissue and bone marrow exhibit different capability to suppress peripheral blood B, natural killer and T cells

IntroductionThe ability to self-renew, be easily expanded in vitro and differentiate into different mesenchymal tissues, render mesenchymal stem cells (MSCs) an attractive therapeutic method for degenerative diseases. The subsequent discovery of their immunosuppressive ability encouraged clinical trials in graft-versus-host disease and auto-immune diseases. Despite sharing several immunophenotypic characteristics and functional capabilities, the differences between MSCs arising from different tissues are still unclear and the published data are conflicting.MethodsHere, we evaluate the influence of human MSCs derived from umbilical cord matrix (UCM), bone marrow (BM) and adipose tissue (AT), co-cultured with phytohemagglutinin (PHA)-stimulated peripheral blood mononuclear cells (MNC), on T, B and natural killer (NK) cell activation; T and B cells’ ability to acquire lymphoblast characteristics; mRNA expression of interleukin-2 (IL-2), forkhead box P3 (FoxP3), T-bet and GATA binding protein 3 (GATA3), on purified T cells, and tumor necrosis factor-alpha (TNF-α), perforin and granzyme B on purified NK cells.ResultsMSCs derived from all three tissues were able to prevent CD4+ and CD8+ T cell activation and acquisition of lymphoblast characteristics and CD56dim NK cell activation, wherein AT-MSCs showed a stronger inhibitory effect. Moreover, AT-MSCs blocked the T cell activation process in an earlier phase than BM- or UCM-MSCs, yielding a greater proportion of T cells in the non-activated state. Concerning B cells and CD56bright NK cells, UCM-MSCs did not influence either their activation kinetics or PHA-induced lymphoblast characteristics, conversely to BM- and AT-MSCs which displayed an inhibitory effect. Besides, when co-cultured with PHA-stimulated MNC, MSCs seem to promote Treg and Th1 polarization, estimated by the increased expression of FoxP3 and T-bet mRNA within purified activated T cells, and to reduce TNF-α and perforin production by activated NK cells.ConclusionsOverall, UCM-, BM- and AT-derived MSCs hamper T cell, B cell and NK cell-mediated immune response by preventing their acquisition of lymphoblast characteristics, activation and changing the expression profile of proteins with an important role in immune function, except UCM-MSCs showed no inhibitory effect on B cells under these experimental conditions. Despite the similarities between the three types of MSCs evaluated, we detect important differences that should be taken into account when choosing the MSC source for research or therapeutic purposes.

A xenogeneic-free bioreactor system for the clinical-scale expansion of human mesenchymal stem/stromal cells

The large cell doses (>1 × 106 cells/kg) used in clinical trials with mesenchymal stem/stromal cells (MSC) will require an efficient production process. Moreover, monitoring and control of MSC ex‐vivo expansion is critical to provide a safe and reliable cell product. Bioprocess engineering approaches, such as bioreactor technology, offer the adequate tools to develop and optimize a cost‐effective culture system for the rapid expansion of human MSC for cellular therapy. Herein, a xenogeneic (xeno)‐free microcarrier‐based culture system was successfully established for bone marrow (BM) MSC and adipose tissue‐derived stem/stromal cell (ASC) cultivation using a 1L‐scale controlled stirred‐tank bioreactor, allowing the production of (1.1 ± 0.1) × 108 and (4.5 ± 0.2) × 107 cells for BM MSC and ASC, respectively, after 7 days. Additionally, the effect of different percent air saturation values (%Airsat) and feeding regime on the proliferation and metabolism of BM MSC was evaluated. No significant differences in cell growth and metabolic patterns were observed under 20% and 9%Airsat. Also, the three different feeding regimes studied—(i) 25% daily medium renewal, (ii) 25% medium renewal every 2 days, and (iii) fed‐batch addition of concentrated nutrients and growth factors every 2 days—yielded similar cell numbers, and only slight metabolic differences were observed. Moreover, the immunophenotype (positive for CD73, CD90 and CD105 and negative for CD31, CD80 and HLA‐DR) and multilineage differentiative potential of expanded cells were not affected upon bioreactor culture. These results demonstrated the feasibility of expanding human MSC from different sources in a clinically relevant expansion configuration in a controlled microcarrier‐based stirred culture system under xeno‐free conditions. The further optimization of this bioreactor culture system will represent a crucial step towards an efficient GMP‐compliant clinical‐scale MSC production system. Biotechnol. Bioeng. 2014;111: 1116–1127.

Spray characterization: numerical prediction of Sauter mean diameter and droplet size distribution

A simplified equation of the Nukiyama-Tanasawa type for droplet size distribution in sprays is obtained from the synergetic concept of entropy information, assuming spherical droplets and zero and infinity as their limit sizes. The introduction of Sauter mean diameter (SMD) definition in that equation yields a new distribution function dependent solely on SMD, which is calculated from available correlations for pressure-jet and pre-filming airblast atomizers. For plain-jet airblast atomizers a new and dimensionally consistent correlation is determined. Several droplet size distributions are then predicted. Experimental data are compared with predictions of SMD; the agreement is satisfactory.

Differentiation of human umbilical cord matrix mesenchymal stem cells into neural-like progenitor cells and maturation into an oligodendroglial-like lineage.

Mesenchymal stem cells (MSCs) are viewed as safe, readily available and promising adult stem cells, which are currently used in several clinical trials. Additionally, their soluble-factor secretion and multi-lineage differentiation capacities place MSCs in the forefront of stem cell types with expected near-future clinical applications. In the present work MSCs were isolated from the umbilical cord matrix (Whartons jelly) of human umbilical cord samples. The cells were thoroughly characterized and confirmed as bona-fide MSCs, presenting in vitro low generation time, high proliferative and colony-forming unit-fibroblast (CFU-F) capacity, typical MSC immunophenotype and osteogenic, chondrogenic and adipogenic differentiation capacity. The cells were additionally subjected to an oligodendroglial-oriented step-wise differentiation protocol in order to test their neural- and oligodendroglial-like differentiation capacity. The results confirmed the neural-like plasticity of MSCs, and suggested that the cells presented an oligodendroglial-like phenotype throughout the differentiation protocol, in several aspects sharing characteristics common to those of bona-fide oligodendrocyte precursor cells and differentiated oligodendrocytes.

Human mesenchymal stem cells from the umbilical cord matrix: successful isolation and ex vivo expansion using serum-/xeno-free culture media.

Mesenchymal stem cells (MSC) could potentially be applied in therapeutic settings due to their multilineage differentiation ability, immunomodulatory properties, as well as their trophic activity. The umbilical cord matrix (UCM) represents a promising source of MSC for biomedical applications. The number of cells isloated per umbilical cord (UC) unit is limited and ex vivo expansion is imperative in order to reach clinically meaningful cell numbers. The limitations of poorly defined reagents (e.g. fetal bovine serum, which is commonly used as a supplement for human MSC expansion) make the use of serum-/xeno-free conditions mandatory. We demonstrated the feasibility of isolating UCM-MSC by plastic adherence using serum-/xeno-free culture medium following enzymatic digestion of UCs, with a 100% success rate. 2.6 ± 0.21 × 10(5) cells were isolated per UC unit, of which 1.9 ± 0.21 × 10(5) were MSC-like cells expressing CD73, CD90, and CD105. When compared to adult sources (bone marrow-derived MSC and adipose-derived stem/stromal cells), UCM-MSC displayed a similar immunophenotype and similar multilineage differentiation ability, while demonstrating a higher expansion potential (average fold increase of 7.4 for serum-containing culture medium and 11.0 for xeno-free culture medium (P3-P6)). The isolation and expansion of UCM-MSC under defined serum-/xeno-free conditions contributes to safer and more effective MSC cellular products, boosting the usefulness of MSC in cellular therapy and tissue engineering.

Gene delivery to human bone marrow mesenchymal stem cells by microporation

Electroporation has been considered one of the most efficient non-viral based methods to deliver genes regardless of frequently observed high cell mortality. In this study we used a microporation technique to optimise the delivery of plasmid DNA encoding green fluorescence protein (GFP) to human bone marrow mesenchymal stem cells (BM-MSC). Using resuspension buffer (RB) and as low as 1.5 x 10⁵ cells and 1 μg of DNA, we achieved 40% of cells expressing the transgene, with cell recovery and cell viabilities of 85% and 90%, respectively. An increase in DNA amount did not significantly increase the number of transfected cells but clearly reduced cell recovery. A face-centered composite design was used to unveil the conditions giving rise to optimal plasmid delivery efficiencies when using a sucrose based microporation buffer (SBB). The BM-MSC proliferation kinetics were mainly affected by the presence of plasmid and not due to the microporation process itself although no effect was observed on their immunophenotypic characteristics and differentiative potential. Based on the data shown herein microporation demonstrated to be a reliable and efficient method to genetically modify hard-to-transfect cells giving rise to the highest levels of cell survival reported so far along with superior gene delivery efficiencies.

Dynamic cell–cell interactions between cord blood haematopoietic progenitors and the cellular niche are essential for the expansion of CD34+, CD34+CD38− and early lymphoid CD7+ cells

Most clinical applications of haematopoietic stem/progenitor cells (HSCs) would benefit from their ex vivo expansion to obtain a therapeutically significant amount of cells from the available donor samples. We studied the impact of cellular interactions between umbilical cord blood (UCB) haematopoietic cells and bone marrow (BM)‐derived mesenchymal stem cells (MSCs) on the ex vivo expansion and differentiative potential of UCB CD34+‐enriched cells. UCB cells were cultured: (a) directly in contact with BM MSC‐derived stromal layers (contact); (b) separated by a microporous membrane (non‐contact); or (c) without stroma (no stroma). Highly dynamic culture events occurred in HSC‐MSC co‐cultures, involving cell–cell interactions, which preceded HSC expansion. Throughout the time in culture [18 days], total cell expansion was significantly higher in contact (fold increase of 280 ± 37 at day 18) compared to non‐contact (85 ± 25). No significant cell expansion was observed in stroma‐free cultures. CD34+ cell expansion was also clearly favoured by direct contact with BM MSCs (35 ± 5‐ and 7 ± 3‐fold increases at day 18 for contact and non‐contact, respectively). Moreover, a higher percentage of CD34+CD38− cells was consistently maintained during the time in culture under contact (8.1 ± 1.9% at day 18) compared to non‐contact (5.7 ± 1.6%). Importantly, direct cell interaction with BM MSCs significantly enhanced the expansion of early lymphoid CD7+ cells, yielding considerably higher (×3–10) progenitor numbers compared to non‐contact conditions. These results highlight the importance of dynamic cell–cell interactions between UCB HSCs and BM MSCs, towards the maximization of HSC expansion ex vivo to obtain clinically relevant cell numbers for multiple settings, such as BM transplantation or somatic cell gene therapy. Copyright