Concise review: Genomic instability in human stem cells: current status and future challenges.

Genomic instability is recognized as one of the most important hurdles in the expanding field of stem cell-based therapies. In the recent years, an accumulating body of evidence has shown that human stem cells undergo a diverse program of biological changes upon ex vivo cultivation that include numerical and structural chromosomal abnormalities, point mutations, variation of telomere length, and epigenetic instability. As the field moves forward, the growing awareness of the risk factors associated with human genome plasticity strongly advocates for the use of extensive genetic screening as part of a quality control platform to attest to the safety of stem cell-based products. Here we present a timely and comprehensive review that addresses the current status and emerging trends of the field, ultimately underscoring the need to implement new regulatory standards able to streamline the route to therapeutic applications.

Separation technologies for stem cell bioprocessing.

Stem cells have been the focus of an intense research due to their potential in Regenerative Medicine, drug discovery, toxicology studies, as well as for fundamental studies on developmental biology and human disease mechanisms. To fully accomplish this potential, the successful application of separation processes for the isolation and purification of stem cells and stem cell-derived cells is a crucial issue. Although separation methods have been used over the past decades for the isolation and enrichment of hematopoietic stem/progenitor cells for transplantation in hemato-oncological settings, recent achievements in the stem cell field have created new challenges including the need for novel scalable separation processes with a higher resolution and more cost-effective. Important examples are the need for high-resolution methods for the separation of heterogeneous populations of multipotent adult stem cells to study their differential biological features and clinical utility, as well as for the depletion of tumorigenic cells after pluripotent stem cell differentiation. Focusing on these challenges, this review presents a critical assessment of separation processes that have been used in the stem cell field, as well as their current and potential applications. The techniques are grouped according to the fundamental principles that govern cell separation, which are defined by the main physical, biophysical, and affinity properties of cells. A special emphasis is given to novel and promising approaches such as affinity-based methods that take advantage of the use of new ligands (e.g., aptamers, lectins), as well as to novel biophysical-based methods requiring no cell labeling and integrated with microscale technologies.

Initial CD34+ cell-enrichment of cord blood determines hematopoietic stem/progenitor cell yield upon ex vivo expansion.

Since umbilical cord blood (UCB), contains a limited hematopoietic stem/progenitor cells (HSC) number, successful expansion protocols are needed to overcome the hurdles associated with inadequate numbers of HSC collected for transplantation. UCB cultures were performed using a human stromal-based serum-free culture system to evaluate the effect of different initial CD34(+) cell enrichments (Low: 24 ± 1.8%, Medium: 46 ± 2.6%, and High: 91 ± 1.5%) on the culture dynamics and outcome of HSC expansion. By combining PKH tracking dye with CD34(+) and CD34(+) CD90(+) expression, we have identified early activation of CD34 expression on CD34(-) cells in Low and Medium conditions, prior to cell division (35 ± 4.7% and 55 ± 4.1% CD34(+) cells at day 1, respectively), affecting proliferation/cell cycle status and ultimately determining CD34(+)/CD34(+) CD90(+) cell yield (High: 14 ± 1.0/3.5 ± 1.4-fold; Medium: 22 ± 2.0/3.4 ± 1,0-fold; Low: 31 ± 3.0/4.4 ± 1.5-fold) after a 7-day expansion. Considering the potential benefits of using expanded UCB HSC in transplantation, here we quantified in single UCB units, the impact of using one/two immunomagnetic sorting cycles (corresponding to Medium and High initial progenitor content), and the average CD34(+) cell recovery for each strategy, on overall CD34(+) cell expansion. The higher cell recovery upon one sorting cycle lead to higher CD34(+) cell numbers after 7 days of expansion (30 ± 2.0 vs. 13 ± 1.0 × 10(6) cells). In particular, a high (>90%) initial progenitor content was not mandatory to successfully expand HSC, since cell populations with moderate levels of enrichment readily increased CD34 expression ex-vivo, generating higher stem/progenitor cell yields. Overall, our findings stress the importance of establishing a balance between the cell proliferative potential and cell recovery upon purification, towards the efficient and cost-effective expansion of HSC for cellular therapy.

Microcarrier expansion of mouse embryonic stem cell-derived neural stem cells in stirred bioreactors.

Neural stem cells (NSCs) are self-renewing multipotent cells, able to differentiate into the phenotypes present in the central nervous system. Applications of NSCs may include toxicology, fundamental research, or cell therapies. The culture of floating cell clusters, called "neurospheres," is widely used for the propagation of NSC populations in vitro but shows several limitations, which may be circumvented by expansion under adherent conditions. In particular, the derivation of distinct populations of NSCs from embryonic stem cells capable of long-term culture under adherent conditions without losing differentiation potential was recently described. However, the expansion of these cells in agitated bioreactors has not been addressed until now and was the aim of this study. Selected microcarriers were tested under dynamic conditions in spinner flasks. Superior performance was observed with polystyrene beads coated with a recombinant peptide containing the Arg-Gly-Asp (RGD) motif (Pronectin F). After optimization of the culture, a 35-fold increase in cell number was achieved after 6 days. High cellular viability and multipotency were maintained throughout the culture. The study presented here may be the basis for the development of larger scale bioprocesses for expansion of these and other populations of adherent NSCs, either from mouse or human origin.

Scalable Manufacturing of Human Hematopoietic Stem/Progenitor Cells Exploiting a Co-culture Platform with Mesenchymal Stromal Cells.

In the context of hematopoietic cell transplantation, hematopoietic stem/progenitor cells (HSPC) from the umbilical cord blood (UCB) present several advantages compared to adult sources including higher proliferative capacity, abundant availability and ease of collection, non-risk and painless harvesting procedure, and lower risk of graft-versus-host disease. However, the therapeutic utility of UCB HSPC has been limited to pediatric patients due to the low cell frequency per unit of UCB. The development of efficient and cost-effective strategies to generate large numbers of functional UCB HSPC ex vivo would boost all current and future medical uses of these cells. Herein, we describe a scalable serum-free co-culture system for the expansion of UCB-derived CD34+-enriched cells using microcarrier-immobilized human bone marrow-derived mesenchymal stromal cells as feeder cells.

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% O(2) (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 O(2), 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% O(2) 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 O(2) presented higher specific consumption of nutrients, especially early in culture, but with lower specific production of inhibitory metabolites. Moreover, 2% O(2) favored CFU-F expansion, while maintaining BM MSC characteristic immunophenotype and differentiative potential. Our results demonstrated a more efficient BM MSC expansion at 2% O(2), 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.

Hypoxia enhances proliferation of mouse embryonic stem cell-derived neural stem cells.

Neural stem (NS) cells can provide a source of material with potential applications for neural drug testing, developmental studies, or novel treatments for neurodegenerative diseases. Herein, the ex vivo expansion of a model system of mouse embryonic stem (mES) cell-derived NS cells was characterized and optimized, cells being cultivated under adherent conditions. Culture was first optimized in terms of initial cell plating density and oxygen concentration, known to strongly influence brain-derived NS cells. To this end, the growth of cells cultured under hypoxic (2%, 5%, and 10% O(2)) and normoxic (20% O(2)) conditions was compared. The results showed that 2-5% oxygen, without affecting multipotency, led to fold increase values in total cell number about twice higher than observed under 20% oxygen (20-fold vs. 10-fold, respectively) this effect being more pronounced when cells were plated at low density. With an optimal cell density of 10(4) cells/cm(2), the maximum growth rates were 1.9 day(-1) under hypoxia versus 1.7 day(-1) under normoxia. Cell division kinetics analysis by flow cytometry based on PKH67 tracking showed that when cultured in hypoxia, cells are at least one divisional generation ahead compared to normoxia. In terms of cell cycle, a larger population in a quiescent G(0) phase was observed in normoxic conditions. The optimization of NS cell culture performed here represents an important step toward the generation of a large number of neural cells from a reduced initial population, envisaging the potential application of these cells in multiple settings.

Kinetic and metabolic analysis of mouse embryonic stem cell expansion under serum-free conditions.

There is a need for a deeper understanding of the biochemical events affecting embryonic stem (ES) cell culture by analyzing the expansion of mouse ES cells in terms of both cell growth and metabolic kinetics. The influence of the initial cell density on cell expansion was assessed. Concomitantly, the biochemical profile of the culture was evaluated, which allowed measuring the consumption of important substrates, such as glucose and glutamine, and the production of metabolic byproducts, like lactate. The results suggest a more efficient cell metabolism in serum-free conditions and a preferential use of glutaminolysis as an energy source during cell expansion at low seeding densities. This work contributes to the development of fully-controlled bioprocesses to produce relevant numbers of ES cells for cell therapies and high-throughput drug screening.

Conditioned Medium From Azurin-Expressing Human Mesenchymal Stromal Cells Demonstrates Antitumor Activity Against Breast and Lung Cancer Cell Lines.

Recently, cell-based therapies have been explored as a strategy to enhance the specificity of anticancer therapeutic agents. In this perspective, human mesenchymal stromal cells (MSC) hold a promising future as cell delivery systems for anticancer proteins due to their unique biological features. In this study, we engineered human MSC to secrete a human codon-optimized version of azurin (hazu), a bacterial protein that has demonstrated anticancer activity toward different cancer models both in vitro and in vivo. To this end, microporation was used to deliver plasmid DNA encoding azurin into MSC derived from bone marrow (BM) and umbilical cord matrix (UCM), leading to expression and secretion of hazu to the conditioned medium (CM). Engineered hazu-MSC were shown to preserve tumor tropism toward breast (MCF-7) and lung (A549) cancer cell lines, comparable to non-modified MSC. Azurin was detected in the CM of transfected MSC and, upon treatment with hazu-MSC-CM, we observed a decrease in cancer cell proliferation, migration, and invasion, and an increase in cell death for both cancer cell lines. Moreover, expression of azurin caused no changes in MSC expression profile of cytokines relevant in the context of cancer progression, thus suggesting that the antitumoral effects induced by hazu-MSC secretome might be due to the presence of azurin independently. In conclusion, data shown herein indicate that MSC-produced azurin in a CM configuration elicits an anticancer effect.

Stem Cell Bioprocessing and Manufacturing.

The next healthcare revolution will apply regenerative medicines using human cells and tissues [...].

Addressing the Manufacturing Challenges of Cell-Based Therapies.

Exciting developments in the cell therapy field over the last decades have led to an increasing number of clinical trials and the first cell products receiving marketing authorization. In spite of substantial progress in the field, manufacturing of cell-based therapies presents multiple challenges that need to be addressed in order to assure the development of safe, efficacious, and cost-effective cell therapies.The manufacturing process of cell-based therapies generally requires tissue collection, cell isolation, culture and expansion (upstream processing), cell harvest, separation and purification (downstream processing), and, finally, product formulation and storage. Each one of these stages presents significant challenges that have been the focus of study over the years, leading to innovative and groundbreaking technological advances, as discussed throughout this chapter.Delivery of cell-based therapies relies on defining product targets while controlling process variable impact on cellular features. Moreover, commercial viability is a critical issue that has had damaging consequences for some therapies. Implementation of cost-effectiveness measures facilitates healthy process development, potentially being able to influence end product pricing.Although cell-based therapies represent a new level in bioprocessing complexity in every manufacturing stage, they also show unprecedented levels of therapeutic potential, already radically changing the landscape of medical care.

Chondrogenic differentiation of mesenchymal stem/stromal cells on 3D porous poly (ε-caprolactone) scaffolds: Effects of material alkaline treatment and chondroitin sulfate supplementation.

Cartilage defects resultant from trauma or degenerative diseases (e.g., osteoarthritis) can potentially be repaired using tissue engineering (TE) strategies combining progenitor cells, biomaterial scaffolds and bio-physical/chemical cues. This work examines promoting chondrogenic differentiation of human bone marrow mesenchymal stem/stromal cells (BM-MSCs) by combining the effects of modified poly (ε-caprolactone) (PCL) scaffolds hydrophilicity and chondroitin sulfate (CS) supplementation in a hypoxic 5% oxygen atmosphere. 3D-extruded PCL scaffolds, characterized by µCT, featured a 21 mm-1 surface area to volume ratio, 390 µm pore size and approximately 100% pore interconnectivity. Scaffold immersion in sodium hydroxide solutions for different periods of time had major effects in scaffold surface morphology, wettability and mechanical properties, but without improvements on cell adhesion. In-situ chondrogenic differentiation of BM-MSC seeded in 3D-extruded PCL scaffolds resulted in higher cell populations and ECM deposition along all scaffold structure, when chondrogenesis was preceded by an expansion phase. Additionally, CS supplementation during BM-MSC expansion was crucial to enhance aggrecan gene expression, known as a hallmark of chondrogenesis. Overall, this study presents an approach to tailor the wettability and mechanical properties of PCL scaffolds and supports the use of CS-supplementation as a biochemical cue in integrated TE strategies for cartilage regeneration.

Scalable Production of Human Mesenchymal Stromal Cell-Derived Extracellular Vesicles Under Serum-/Xeno-Free Conditions in a Microcarrier-Based Bioreactor Culture System.

Mesenchymal stromal cells (MSC) hold great promise for tissue engineering and cell-based therapies due to their multilineage differentiation potential and intrinsic immunomodulatory and trophic activities. Over the past years, increasing evidence has proposed extracellular vesicles (EVs) as mediators of many of the MSC-associated therapeutic features. EVs have emerged as mediators of intercellular communication, being associated with multiple physiological processes, but also in the pathogenesis of several diseases. EVs are derived from cell membranes, allowing high biocompatibility to target cells, while their small size makes them ideal candidates to cross biological barriers. Despite the promising potential of EVs for therapeutic applications, robust manufacturing processes that would increase the consistency and scalability of EV production are still lacking. In this work, EVs were produced by MSC isolated from different human tissue sources [bone marrow (BM), adipose tissue (AT), and umbilical cord matrix (UCM)]. A serum-/xeno-free microcarrier-based culture system was implemented in a Vertical-WheelTM bioreactor (VWBR), employing a human platelet lysate culture supplement (UltraGROTM-PURE), toward the scalable production of MSC-derived EVs (MSC-EVs). The morphology and structure of the manufactured EVs were assessed by atomic force microscopy, while EV protein markers were successfully identified in EVs by Western blot, and EV surface charge was maintained relatively constant (between -15.5 ± 1.6 mV and -19.4 ± 1.4 mV), as determined by zeta potential measurements. When compared to traditional culture systems under static conditions (T-flasks), the VWBR system allowed the production of EVs at higher concentration (i.e., EV concentration in the conditioned medium) (5.7-fold increase overall) and productivity (i.e., amount of EVs generated per cell) (3-fold increase overall). BM, AT and UCM MSC cultured in the VWBR system yielded an average of 2.8 ± 0.1 × 1011, 3.1 ± 1.3 × 1011, and 4.1 ± 1.7 × 1011 EV particles (n = 3), respectively, in a 60 mL final volume. This bioreactor system also allowed to obtain a more robust MSC-EV production, regarding their purity, compared to static culture. Overall, we demonstrate that this scalable culture system can robustly manufacture EVs from MSC derived from different tissue sources, toward the development of novel therapeutic products.

Differences amid bone marrow and cord blood hematopoietic stem/progenitor cell division kinetics.

Human hematopoietic stem/progenitor cells (HSC) isolated based upon specific patterns of CD34 and CD38 expression, despite phenotypically identical, were found to be functionally heterogeneous, raising the possibility that reversible expression of these antigens may occur during cellular activation and/or proliferation. In these studies, we combined PKH67 tracking with CD34/CD38 immunostaining to compare cell division kinetics between human bone marrow (BM) and cord blood (CB)-derived HSC expanded in a serum-free/stromal-based system for 14 days (d), and correlated CD34 and CD38 expression with the cell divisional history. CB cells began dividing 24 h earlier than BM cells, and significantly higher numbers underwent mitosis during the time in culture. By d10, over 55% of the CB-cells reached the ninth generation, whereas BM-cells were mostly distributed between the fifth and seventh generation. By d14, all CB cells had undergone multiple cell divisions, while 0.7-3.8% of BM CD34(+) cells remained quiescent. Furthermore, the percentage of BM cells expressing CD34 decreased from 60.8 +/- 6.3% to 30.6 +/- 6.7% prior to initiating division, suggesting that downmodulation of this antigen occurred before commencement of proliferation. Moreover, with BM, all primitive CD34(+)CD38(-) cells present at the end of culture arose from proliferating CD34(+)CD38(+) cells that downregulated CD38 expression, while in CB, a CD34(+)CD38(-) population was maintained throughout culture. These studies show that BM and CB cells differ significantly in cell division kinetics and expression of CD34 and CD38, and that the inherent modulation of these antigens during ex vivo expansion may lead to erroneous quantification of the stem cell content of the expanded graft.

A human stromal-based serum-free culture system supports the ex vivo expansion/maintenance of bone marrow and cord blood hematopoietic stem/progenitor cells.

OBJECTIVE: We investigated the role of human stromal layers (hu-ST) on the ex vivo expansion/maintenance of human hematopoietic stem/progenitor cells (HSC) from adult bone marrow (BM) and umbilical cord blood (CB). MATERIALS AND METHODS: BM and CB CD34(+)-enriched cells were cultured in serum-free medium supplemented with SCF, bFGF, LIF, and Flt-3, in the presence or absence of stroma, and analyzed for proliferation, phenotype, and clonogenic potential. RESULTS: Significant expansion of BM and CB CD34(+) and CD34(+)CD38(-) cells were achieved in the presence of hu-ST. The differentiative potential of both BM and CB CD34(+)-enriched cells cocultured with hu-ST was primarily shifted toward the myeloid lineage, while maintaining/expanding a CD7(+) population. Clonogenic analysis of the expanded cells showed increases in progenitors of the myeloid lineage, including colony-forming unit-granulocyte, macrophage (CFU-GM) and colony-forming unit-granulocyte, erythroid, macrophage, megakaryocyte (CFU-Mix) for both BM (stroma and stroma-free conditions) and CB cells in the presence of stroma. CONCLUSIONS: These results indicate that adult hu-ST in the presence of appropriate cytokines can be used to efficiently expand/maintain myeloid and lymphoid cell populations from human BM and CB HSC.

Clinical-Grade Manufacturing of Therapeutic Human Mesenchymal Stem/Stromal Cells in Microcarrier-Based Culture Systems.

The therapeutic potential of mesenchymal stem/stromal cells (MSC) has triggered the need for high cell doses in a vast number of clinical applications. This demand requires the development of good manufacturing practices (GMP)-compliant ex vivo expansion protocols that should be effective to deliver a robust and reproducible supply of clinical-grade cells in a safe and cost-effective manner. Controlled stirred-tank bioreactor systems under xenogeneic (xeno)-free culture conditions offer ideal settings to develop and optimize cell manufacturing to meet the standards and needs of human MSC for cellular therapies. Herein we describe two microcarrier-based stirred culture systems using spinner flasks and controlled stirred-tank bioreactors under xeno-free conditions for the efficient ex vivo expansion of human bone marrow and adipose tissue-derived MSC.

Scalable ex vivo expansion of human mesenchymal stem/stromal cells in microcarrier-based stirred culture systems.

The clinical demand for human mesenchymal stem/stromal cells (MSC) drives the need for reproducible, cost-effective, and good manufacturing practices (GMP)-compliant ex vivo expansion protocols. Bioprocess engineering strategies, namely controlled stirred bioreactor systems combined with the use of xenogeneic(xeno)-free materials, provide proper tools to develop and optimize cell manufacturing for the rapid expansion of human MSC for cellular therapies. Herein we describe a microcarrier-based stirred culture system operating under xeno-free conditions using a controlled stirred-tank bioreactor for an efficient and controlled ex vivo expansion of human MSC. This culture platform can be applied to MSC from different human sources, as well as different microcarriers and xeno-free medium formulations.

A xeno-free microcarrier-based stirred culture system for the scalable expansion of human mesenchymal stem/stromal cells isolated from bone marrow and adipose tissue.

Human mesenchymal stem/stromal cells (MSC) are promising candidates for cell-based therapies and the development of microcarrier-based cultures in scalable bioreactors with well-defined xenogeneic-free components represent important milestones towards the clinical-scale production of these cells. In this work, we optimized our previously developed xeno-free microcarrier-based system for the scalable expansion of human MSC isolated from bone marrow (BM MSC) and adipose-derived stem/stromal cells (ASC). By adapting the agitation/feeding protocol at the initial cell seeding/cultivation stage in spinner flasks, we were able to maximize cell expansion rate and final cell yield. Maximal cell densities of 3.6 × 10(5) and 1.9 × 10(5) cells/mL were obtained for BM MSC (0.60 ± 0.04 day(-1) ) and ASC (0.9 ± 0.1 day(-1) ) cultures, upon seven and eight days of cultivation, respectively. Ready-to-use microcarriers Synthemax® II and Enhanced Attachment® supported identical expansion performance of BM MSC, turning those effective alternatives to the pre-coated plastic microcarriers used in our xeno-free scalable culture system. Importantly, expanded MSC maintained their immunophenotype and multilineage differentiation potential. Moreover, secretome analysis suggested a priming effect of stirred culture conditions on cytokine production by MSC. This culture system yielded considerable final cell densities that can be scaled-up to controlled large-scale bioreactors allowing a more efficient, safe and cost-effective MSC production for clinical settings.

Hematopoietic stem cells: from the bone to the bioreactor.

The ex vivo expansion of human hematopoietic stem cells is a rapidly developing area with a broad range of biomedical applications. The mechanisms of renewal, differentiation and plasticity of stem cells are currently under intense investigation. However, the complexity of hematopoiesis, the heterogeneity of the culture population and the complex interplay between the culture parameters that significantly influence the proliferation and differentiation of hematopoietic cells have impaired the translation of small scale results to the highly demanded large-scale applications. The better understanding of these mechanisms is providing the basis for more rational approaches to the ex vivo expansion of hematopoietic stem cells. Efforts are now being made to establish a rational design of bioreactor systems, allowing the modeling and control of large-scale production of stem cells and the study of their proliferation and differentiation, under conditions as similar as possible to those in vivo.