Maroof M. Adil

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

Summary 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.

Efficient generation of hPSC-derived midbrain dopaminergic neurons in a fully defined, scalable, 3D biomaterial platform

Pluripotent stem cells (PSCs) have major potential as an unlimited source of functional cells for many biomedical applications; however, the development of cell manufacturing systems to enable this promise faces many challenges. For example, there have been major recent advances in the generation of midbrain dopaminergic (mDA) neurons from stem cells for Parkinson’s Disease (PD) therapy; however, production of these cells typically involves undefined components and difficult to scale 2D culture formats. Here, we used a fully defined, 3D, thermoresponsive biomaterial platform to rapidly generate large numbers of action-potential firing mDA neurons after 25 days of differentiation (~40% tyrosine hydroxylase (TH) positive, maturing into 25% cells exhibiting mDA neuron-like spiking behavior). Importantly, mDA neurons generated in 3D exhibited a 30-fold increase in viability upon implantation into rat striatum compared to neurons generated on 2D, consistent with the elevated expression of survival markers FOXA2 and EN1 in 3D. A defined, scalable, and resource-efficient cell culture platform can thus rapidly generate high quality differentiated cells, both neurons and potentially other cell types, with strong potential to accelerate both basic and translational research.

A Rationally Designed, General Strategy for Membrane Orientation of Photoinduced Electron Transfer-Based Voltage-Sensitive Dyes

Voltage imaging with fluorescent dyes offers promise for interrogating the complex roles of membrane potential in coordinating the activity of neurons in the brain. Yet, low sensitivity often limits the broad applicability of optical voltage indicators. In this paper, we use molecular dynamics (MD) simulations to guide the design of new, ultrasensitive fluorescent voltage indicators that use photoinduced electron transfer (PeT) as a voltage-sensing switch. MD simulations predict an approximately 16% increase in voltage sensitivity resulting purely from improved alignment of dye with the membrane. We confirm this theoretical finding by synthesizing 9 new voltage-sensitive (VoltageFluor, or VF) dyes and establishing that all of them display the expected improvement of approximately 19%. This synergistic outworking of theory and experiment enabled computational and theoretical estimation of VF dye orientation in lipid bilayers and has yielded the most sensitive PeT-based VF dye to date. We use this new voltage indicator to monitor voltage spikes in neurons from rat hippocampus and human pluripotent-stem-cell-derived dopaminergic neurons.

cAMP and EPAC Signaling Functionally Replace OCT4 During Induced Pluripotent Stem Cell Reprogramming

The advent of induced pluripotent stem cells--generated via the ectopic overexpression of reprogramming factors such as OCT4, SOX2, KLF4, and C-MYC (OSKM) in a differentiated cell type--has enabled groundbreaking research efforts in regenerative medicine, disease modeling, and drug discovery. Although initial studies have focused on the roles of nuclear factors, increasing evidence highlights the importance of signal transduction during reprogramming. By utilizing a quantitative, medium-throughput screen to initially identify signaling pathways that could potentially replace individual transcription factors during reprogramming, we initially found that several pathways--such as Notch, Smoothened, and cyclic AMP (cAMP) signaling--were capable of generating alkaline phosphatase positive colonies in the absence of OCT4, the most stringently required Yamanaka factor. After further investigation, we discovered that cAMP signal activation could functionally replace OCT4 to induce pluripotency, and results indicate that the downstream exchange protein directly activated by cAMP (EPAC) signaling pathway rather than protein kinase A (PKA) signaling is necessary and sufficient for this function. cAMP signaling may reduce barriers to reprogramming by contributing to downstream epithelial gene expression, decreasing mesenchymal gene expression, and increasing proliferation. Ultimately, these results elucidate mechanisms that could lead to new reprogramming methodologies and advance our understanding of stem cell biology.

Thermoreversible Hyaluronic Acid-PNIPAAm Hydrogel Systems for 3D Stem Cell Culture

Human pluripotent stem cells (hPSCs) offer considerable potential for biomedical applications including drug screening and cell replacement therapies. Clinical translation of hPSCs requires large quantities of high quality cells, so scalable methods for cell culture are needed. However, current methods are limited by scalability, the use of animal-derived components, and/or low expansion rates. A thermoresponsive 3D hydrogel for scalable hPSC expansion and differentiation into several defined lineages is recently reported. This system would benefit from increased control over material properties to further tune hPSC behavior, and here a scalable 3D biomaterial with the capacity to tune both the chemical and the mechanical properties is demonstrated to promote hPSC expansion under defined conditions. This 3D biomaterial, comprised of hyaluronic acid and poly(N-isopropolyacrylamide), has thermoresponsive properties that readily enable mixing with cells at low temperatures, physical encapsulation within the hydrogel upon elevation at 37 °C, and cell recovery upon cooling and reliquefaction. After optimization, the resulting biomaterial supports hPSC expansion over long cell culture periods while maintaining cell pluripotency. The capacity to modulate the mechanical and chemical properties of the hydrogel provides a new avenue to expand hPSCs for future therapeutic application.

hPSC-Derived Striatal Cells Generated Using a Scalable 3D Hydrogel Promote Recovery in a Huntington Disease Mouse Model

Summary Huntington disease (HD) is an inherited, progressive neurological disorder characterized by degenerating striatal medium spiny neurons (MSNs). One promising approach for treating HD is cell replacement therapy, where lost cells are replaced by MSN progenitors derived from human pluripotent stem cells (hPSCs). While there has been remarkable progress in generating hPSC-derived MSNs, current production methods rely on two-dimensional culture systems that can include poorly defined components, limit scalability, and yield differing preclinical results. To facilitate clinical translation, here, we generated striatal progenitors from hPSCs within a fully defined and scalable PNIPAAm-PEG three-dimensional (3D) hydrogel. Transplantation of 3D-derived striatal progenitors into a transgenic mouse model of HD slowed disease progression, improved motor coordination, and increased survival. In addition, the transplanted cells developed an MSN-like phenotype and formed synaptic connections with host cells. Our results illustrate the potential of scalable 3D biomaterials for generating striatal progenitors for HD cell therapy.

Cheaper and less variable expansion

Substituting growth factors with small molecules in the culture medium for the expansion of human pluripotent stem cells reduces costs and lot-to-lot variability.