Proteomics of novel induced pluripotent stem cell-derived vascular endothelial cells reveal extensive similarity with an immortalized human endothelial cell line.

The vascular endothelium constitutes the inner lining of the blood vessel, and malfunction and injuries of the endothelium can cause cardiovascular diseases as well as other diseases including stroke, tumor growth, and chronic kidney failure. Generation of effective sources to replace injured endothelial cells (ECs) could have significant clinical impact, and somatic cell sources like peripheral or cord blood cannot credibly supply enough endothelial cell progenitors for multitude of treatments. Pluripotent stem cells are a promising source for a reliable EC supply, which have the potential to restore tissue function and treat vascular diseases. We have developed methods to differentiate induced pluripotent stem cells (iPSCs) efficiently and robustly across multiple iPSC lines into nontissue-specific pan vascular ECs (iECs) with high purity. These iECs present with canonical endothelial cell markers and exhibit measures of endothelial cell functionality with the uptake of Dil fluorescent dye-labeled acetylated low-density lipoprotein (Dil-Ac-LDL) and tube formation. Using proteomic analysis, we revealed that the iECs are more proteomically similar to established human umbilical vein ECs (HUVECs) than to iPSCs. Posttranslational modifications (PTMs) were most shared between HUVECs and iECs, and potential targets for increasing the proteomic similarity of iECs to HUVECs were identified. Here we demonstrate an efficient robust method to differentiate iPSCs into functional ECs, and for the first time provide a comprehensive protein expression profile of iECs, which indicates their similarities with a widely used immortalized HUVECs, allowing for further mechanistic studies of EC development, signaling, and metabolism for future regenerative applications.NEW & NOTEWORTHY We have developed methods to differentiate induced pluripotent stem cells (iPSCs) across multiple iPSC lines into nontissue-specific pan vascular ECs (iECs) and demonstrated the proteomic similarity of these cells to a widely used endothelial cell line (HUVECs). We also identified posttranslational modifications and targets for increasing the proteomic similarity of iECs to HUVECs. In the future, iECs can be used to study EC development, signaling, and metabolism for future regenerative applications.

Cell transplantation strategies for acquired and inherited disorders of peripheral myelin.

Objective: To investigate transplantation of rat Schwann cells or human iPSC-derived neural crest cells and derivatives into models of acquired and inherited peripheral myelin damage. Methods: Primary cultured rat Schwann cells labeled with a fluorescent protein for monitoring at various times after transplantation. Human-induced pluripotent stem cells (iPSCs) were differentiated into neural crest stem cells, and subsequently toward a Schwann cell lineage via two different protocols. Cell types were characterized using flow cytometry, immunocytochemistry, and transcriptomics. Rat Schwann cells and human iPSC derivatives were transplanted into (1) nude rats pretreated with lysolecithin to induce demyelination or (2) a transgenic rat model of dysmyelination due to PMP22 overexpression. Results: Rat Schwann cells transplanted into sciatic nerves with either toxic demyelination or genetic dysmyelination engrafted successfully, and migrated longitudinally for relatively long distances, with more limited axial migration. Transplanted Schwann cells engaged existing axons and displaced dysfunctional Schwann cells to form normal-appearing myelin. Human iPSC-derived neural crest stem cells and their derivatives shared similar engraftment and migration characteristics to rat Schwann cells after transplantation, but did not further differentiate into Schwann cells or form myelin. Interpretation: These results indicate that cultured Schwann cells surgically delivered to peripheral nerve can engraft and form myelin in either acquired or inherited myelin injury, as proof of concept for pursuing cell therapy for diseases of peripheral nerve. However, lack of reliable technology for generating human iPSC-derived Schwann cells for transplantation therapy remains a barrier in the field.

Label-free quantitative proteomic profiling identifies disruption of ubiquitin homeostasis as a key driver of Schwann cell defects in spinal muscular atrophy.

Low levels of survival of motor neuron (SMN) protein cause the neuromuscular disease spinal muscular atrophy (SMA), characterized by degeneration of lower motor neurons and atrophy of skeletal muscle. Recent work demonstrated that low levels of SMN also trigger pathological changes in Schwann cells, leading to abnormal axon myelination and disrupted deposition of extracellular matrix proteins in peripheral nerve. However, the molecular pathways linking SMN depletion to intrinsic defects in Schwann cells remained unclear. Label-free proteomics analysis of Schwann cells isolated from SMA mouse peripheral nerve revealed widespread changes to the Schwann cell proteome, including disruption to growth/proliferation, cell death/survival, and molecular transport pathways. Functional clustering analyses revealed significant disruption to a number of proteins contributing to ubiquitination pathways, including reduced levels of ubiquitin-like modifier activating enzyme 1 (Uba1). Pharmacological suppression of Uba1 in Schwann cells was sufficient to reproduce the defective myelination phenotype seen in SMA. These findings demonstrate an important role for SMN protein and ubiquitin-dependent pathways in maintaining Schwann cell homeostasis and provide significant additional experimental evidence supporting a key role for ubiquitin pathways and, Uba1 in particular, in driving SMA pathogenesis across a broad range of cells and tissues.

SMN-dependent intrinsic defects in Schwann cells in mouse models of spinal muscular atrophy.

Low levels of survival of motor neuron (SMN) protein lead to spinal muscular atrophy (SMA). The major pathological hallmark of SMA is a loss of lower motor neurons from spinal cord and peripheral nerve. However, recent studies have revealed pathological changes in other cells and tissues of the neuromuscular system. Here, we demonstrate intrinsic, SMN-dependent defects in Schwann cells in SMA. Myelination in intercostal nerves was perturbed at early- and late-symptomatic stages of disease in two mouse models of SMA. Similarly, maturation of axo-glial interactions at paranodes was disrupted in SMA mice. In contrast, myelination of motor axons in the corticospinal tract of the spinal cord occurred normally. Schwann cells isolated from SMA mice had significantly reduced levels of SMN and failed to express key myelin proteins following differentiation, likely due to perturbations in protein translation and/or stability rather than transcriptional defects. Myelin protein expression was restored in SMA Schwann cells following transfection with an SMN construct. Co-cultures of healthy neurons with diseased Schwann cells revealed deficient myelination, suggestive of intrinsic defects in Schwann cells, as well as reduced neurite stability. Alongside myelination defects, SMA Schwann cells failed to express normal levels of key extracellular matrix proteins, including laminin α2. We conclude that Schwann cells require high levels of SMN protein for their normal development and function in vivo, with reduced levels of SMN resulting in myelination defects, delayed maturation of axo-glial interactions and abnormal composition of extracellular matrix in peripheral nerve.