Defined and Scalable Differentiation of Human Oligodendrocyte Precursors from Pluripotent Stem Cells in a 3D Culture System.

Oligodendrocyte precursor cells (OPCs) offer considerable potential for the treatment of demyelinating diseases and injuries of the CNS. However, generating large quantities of high-quality OPCs remains a substantial challenge that impedes their therapeutic application. Here, we show that OPCs can be generated from human pluripotent stem cells (hPSCs) in a three-dimensional (3D), scalable, and fully defined thermoresponsive biomaterial system. We used CRISPR/Cas9 to create a NKX2.2-EGFP human embryonic stem cell reporter line that enabled fine-tuning of early OPC specification and identification of conditions that markedly increased the number of OLIG2+ and NKX2.2+ cells generated from hPSCs. Transplantation of 50-day-old OPCs into the brains of NOD/SCID mice revealed that progenitors generated in 3D without cell selection or purification subsequently engrafted, migrated, and matured into myelinating oligodendrocytes in vivo. These results demonstrate the potential of harnessing lineage reporter lines to develop 3D platforms for rapid and large-scale production of OPCs.

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.

Clinical-scale purification of pluripotent stem cell derivatives for cell-based therapies.

Human pluripotent stem cells (hPSCs) have the potential to revolutionize cell-replacement therapies because of their ability to self renew and differentiate into nearly every cell type in the body. However, safety concerns have delayed the clinical translation of this technology. One cause for this is the capacity that hPSCs have to generate tumors after transplantation. Because of the challenges associated with achieving complete differentiation into clinically relevant cell types, the development of safe and efficient strategies for purifying committed cells is essential for advancing hPSC-based therapies. Several purification strategies have now succeeded in generating non-tumorigenic and homogeneous cell-populations. These techniques typically enrich for cells by either depleting early committed populations from teratoma-initiating hPSCs or by positively selecting cells after differentiation. Here we review the working principles behind separation methods that have facilitated the safe and controlled application of hPSC-derived cells in laboratory settings and pre-clinical research. We underscore the need for improving and integrating purification strategies within differentiation protocols in order to unlock the therapeutic potential of hPSCs.

Integrated platform for production and purification of human pluripotent stem cell-derived neural precursors.

Human pluripotent stem cells (hPSCs) are a promising source of cells for clinical applications, such as transplantation of clinically engineered tissues and organs, and drug discovery programs due to their ability to self-renew and to be differentiated into cells from the three embryonic germ layers. In this study, the differentiation of two hPSC-lines into neural precursors (NPs) was accomplished with more than 80% efficiency, by means of the dual-SMAD inhibition protocol, based on the use of two small molecules (SB431542 and LDN193189) to generate Pax6 and Nestin-positive neural entities. One of the major hurdles related to the in vitro generation of PSC-derived populations is the tumorigenic potential of cells that remain undifferentiated. These remaining hPSCs have the potential to generate teratomas after being transplanted, and may interfere with the outcome of in vitro differentiation protocols. One strategy to tackle this problem is to deplete these "contaminating" cells during the differentiation process. Magnetic activated cell sorting (MACS) was used for the first time for purification of hPSC-derived NPs after the neural commitment stage using anti-Tra-1-60 micro beads for negative selection of the unwanted hPSCs. The depletion had an average efficiency of 80.4 ± 5% and less than 1.5% of Tra-1-60 positive cells were present in the purified populations. After re-plating, the purified neural precursors maintained their phenotype, and the success of the preparative purification with MACS was further confirmed with a decrease of 94.3% in the number of Oct4-positive proliferating hPSC colonies. Thus, the integration of the MACS depletion step with the neural commitment protocol paves the way towards the establishment of a novel bioprocess for production of purified populations of hPSC-derived neural cells for different 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.

New insights into the mechanisms of embryonic stem cell self-renewal under hypoxia: a multifactorial analysis approach.

Previous reports have shown that culturing mouse embryonic stem (mES) cells at different oxygen tensions originated different cell proliferation patterns and commitment stages depending on which signaling pathways are activated or inhibited to support the pluripotency state. Herein we provide new insights into the mechanisms by which oxygen is influencing mES cell self-renewal and pluripotency. A multifactorial approach was developed to rationally evaluate the singular and interactive control of MEK/ERK pathway, GSK-3 inhibition, and LIF/STAT3 signaling at physiological and non-physiological oxygen tensions. Collectively, our methodology revealed a significant role of GSK-3-mediated signaling towards maintenance of mES cell pluripotency at lower O(2) tensions. Given the central role of this signaling pathway, future studies will need to focus on the downstream mechanisms involved in ES cell self-renewal under such conditions, and ultimately how these findings impact human models of pluripotency.

Plasmid DNA size does affect nonviral gene delivery efficiency in stem cells.

Genetic modification of stem cells, prior to transplantation, can enhance their survival and can improve their function in cell therapy settings. Mesenchymal stem cells (MSC) are considered one of the most promising tools for cell-based gene therapy, due to their multipotency, ease of isolation, as well as their high ex vivo expansion potential. Neural stem cells (NSC) may also present an ideal route for gene therapy and have been considered for use in cell replacement therapies in various neurodegenerative diseases. Gene therapy-based applications require the transfer of genetic material, either by viral or nonviral gene delivery methods, although the latter has been associated with low efficiencies, especially within hard to transfect cells as stem cells. Herein, we present results on the influence of plasmid size in gene delivery to human MSC and mouse NSC. We used minimized plasmids encoding a fluorescent protein but lacking the antibiotic resistance gene. This work shows that (1) for smaller plasmids the intracellular plasmid copy number can be up to 2.6-fold higher, and (2) the number of cells presenting fluorescence can be twice the number obtained for larger plasmids. Furthermore, by using plasmid constructs containing different polyA signals, we also demonstrated that differences between the plasmids depend largely on the transgene mRNA level. Based on our data we demonstrate that plasmid size severely affects the efficiency of nuclear uptake and we propose that it can also affect the rate of heterochromatin associated gene silencing in stem cells.

Different stages of pluripotency determine distinct patterns of proliferation, metabolism, and lineage commitment of embryonic stem cells under hypoxia.

Oxygen tension is an important component of the stem cell microenvironment. Herein, we have studied the effect of low oxygen levels (2% O(2)), or hypoxia, in the expansion of mouse embryonic stem (ES) cells. In the presence of leukemia inhibitory factor (LIF), cell proliferation was reduced under hypoxia and a simultaneous reduction in cell viability was also observed. Morphological changes and different cell cycle patterns were observed, suggesting some early differentiation under hypoxic conditions. However, when cells were maintained in a ground state of pluripotency, by inhibition of autocrine FGF4/ERK and GSK3 signaling, hypoxia did not affect cell proliferation, and did not induce early differentiation. As expected, there was an increase in lactate-specific production rate and a significant increase in the glucose consumption under hypoxic conditions. Nevertheless, during neural commitment, low oxygen tension exerted a positive effect on early differentiation of ground-state ES cells, resulting in a faster commitment toward neural progenitors. Overall our results demonstrate the need to specifically regulate the oxygen content, especially hypoxia, along with other culture conditions, when developing new strategies for ES cell expansion and/or controlled differentiation.

Three-dimensional cell culture microarray for high-throughput studies of stem cell fate.

We have developed a novel three-dimensional (3D) cellular microarray platform to enable the rapid and efficient tracking of stem cell fate and quantification of specific stem cell markers. This platform consists of a miniaturized 3D cell culture array on a functionalized glass slide for spatially addressable high-throughput screening. A microarray spotter was used to deposit cells onto a modified glass surface to yield an array consisting of cells encapsulated in alginate gel spots with volumes as low as 60 nL. A method based on an immunofluorescence technique scaled down to function on a cellular microarray was also used to quantify specific cell marker protein levels in situ. Our results revealed that this platform is suitable for studying the expansion of mouse embryonic stem (ES) cells as they retain their pluripotent and undifferentiated state. We also examined neural commitment of mouse ES cells on the microarray and observed the generation of neuroectodermal precursor cells characterized by expression of the neural marker Sox-1, whose levels were also measured in situ using a GFP reporter system. In addition, the high-throughput capacity of the platform was tested using a dual-slide system that allowed rapid screening of the effects of tretinoin and fibroblast growth factor-4 (FGF-4) on the pluripotency of mouse ES cells. This high-throughput platform is a powerful new tool for investigating cellular mechanisms involved in stem cell expansion and differentiation and provides the basis for rapid identification of signals and conditions that can be used to direct cellular responses.