Xeno-free induced pluripotent stem cell-derived neural progenitor cells for in vivo applications.

BACKGROUND: Currently, there is no regenerative therapy for patients with neurological and neurodegenerative disorders. Cell-therapies have emerged as a potential treatment for numerous brain diseases. Despite recent advances in stem cell technology, major concerns have been raised regarding the feasibility and safety of cell therapies for clinical applications. METHODS: We generated good manufacturing practice (GMP)-compatible neural progenitor cells (NPCs) from transgene- and xeno-free induced pluripotent stem cells (iPSCs) that can be smoothly adapted for clinical applications. NPCs were characterized in vitro for their differentiation potential and in vivo after transplantation into wild type as well as genetically immunosuppressed mice. RESULTS: Generated NPCs had a stable gene-expression over at least 15 passages and could be scaled for up to 1018 cells per initially seeded 106 cells. After withdrawal of growth factors in vitro, cells adapted a neural fate and mainly differentiated into active neurons. To ensure a pure NPC population for in vivo applications, we reduced the risk of iPSC contamination by applying micro RNA-switch technology as a safety checkpoint. Using lentiviral transduction with a fluorescent and bioluminescent dual-reporter construct, combined with non-invasive in vivo bioluminescent imaging, we longitudinally tracked the grafted cells in healthy wild-type and genetically immunosuppressed mice as well as in a mouse model of ischemic stroke. Long term in-depth characterization revealed that transplanted NPCs have the capability to survive and spontaneously differentiate into functional and mature neurons throughout a time course of a month, while no residual pluripotent cells were detectable. CONCLUSION: We describe the generation of transgene- and xeno-free NPCs. This simple differentiation protocol combined with the ability of in vivo cell tracking presents a valuable tool to develop safe and effective cell therapies for various brain injuries.

Intracerebral Transplantation and In Vivo Bioluminescence Tracking of Human Neural Progenitor Cells in the Mouse Brain.

Cell therapy has long been an emerging treatment paradigm in experimental neurobiology. However, cell transplantation studies often rely on end-point measurements and can therefore only evaluate longitudinal changes of cell migration and survival to a limited extent. This paper provides a reliable, minimally invasive protocol to transplant and longitudinally track neural progenitor cells (NPCs) in the adult mouse brain. Before transplantation, cells are transduced with a lentiviral vector comprising a bioluminescent (firefly-luciferase) and fluorescent (green fluorescent protein [GFP]) reporter. The NPCs are transplanted into the right cortical hemisphere using stereotaxic injections in the sensorimotor cortex. Following transplantation, grafted cells were detected through the intact skull for up to five weeks (at days 0, 3, 14, 21, 35) with a resolution limit of 6,000 cells using in vivo bioluminescence imaging. Subsequently, the transplanted cells are identified in histological brain sections and further characterized with immunofluorescence. Thus, this protocol provides a valuable tool to transplant, track, quantify, and characterize cells in the mouse brain.

Heterogeneous expression of ACE2 and TMPRRS2 in mesenchymal stromal cells.

The outbreak of COVID-19 has become a serious public health emergency. The virus targets cells by binding the ACE2 receptor. After infection, the virus triggers in some humans an immune storm containing the release of proinflammatory cytokines and chemokines followed by multiple organ failure. Several vaccines are enrolled, but an effective treatment is still missing. Mesenchymal stem cells (MSCs) have shown to secrete immunomodulatory factors that suppress this cytokine storm. Therefore, MSCs have been suggested as a potential treatment option for COVID-19. We report here that the ACE2 expression is minimal or nonexistent in MSC derived from three different human tissue sources (adipose tissue, umbilical cord Wharton`s jelly and bone marrow). In contrast, TMPRSS2 that is implicated in SARS-CoV-2 entry has been detected in all MSC samples. These results are of particular importance for future MSC-based cell therapies to treat severe cases after COVID-19 infection.

High Frequency of Tumor Propagating Cells in Fusion-Positive Rhabdomyosarcoma.

Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in children. Fusion-positive RMS (FPRMS), expressing the PAX3/7-FOXO1, has a worse prognosis compared to the more common fusion-negative RMS (FNRMS). Although several studies reported hierarchical organization for FNRMS with the identification of cancer stem cells, the cellular organization of FPRMS is not yet clear. In this study we investigated the expression of key stem cell markers, developed a sphere assay, and investigated the seven most common FPRMS cell lines for subpopulations of tumor propagating cancer stem-like cells, also called cancer stem cells (CSCs). Moreover, loss- and gain-of-functions of the stem cell genes SOX2, OCT4, and NANOG were investigated in the same cells. Single-cell clonal analysis was performed in vitro as well as in vivo. We found that no stable CSC subpopulation could be enriched in FPRMS. Unlike depletion of PAX3-FOXO1, neither overexpression nor siRNA-mediated downregulation of SOX2, OCT4, and NANOG affected physiology of RMS cells. Every single subclone-derived cell clone initiated tumor growth in mice, despite displaying considerable heterogeneity in gene expression. FPRMS appears to contain a high frequency of tumor propagating stem-like cells, which could explain their higher propensity for metastasis and relapse. Their dependency on PAX3-FOXO1 activity reinforces the importance of the fusion protein as the key therapeutic target.

Increased maturation of iPSC-derived neurons in a hydrogel-based 3D culture.

BACKGROUND: Induced pluripotent stem cells (iPSCs) can be differentiated into virtually every desired cell type, offering significant potential for modeling human diseases in vitro. A disadvantage is that iPSC-derived cells represent an immature, which presents a major limitation for modeling age-related diseases such as Alzheimer's disease. Evidence suggests that culturing iPSC neurons in a 3D environment may increase neuronal maturity. However, current 3D cell culture systems are cumbersome and time-consuming. NEW METHOD: We cultured iPSC-derived excitatory neurons in 3D precast hydrogel plates and compared their maturation to 2D monolayer cultures. COMPARISON WITH EXISTING METHODS: In contrast to other hydrogel-based 3D culture techniques, which require full encapsulation of cells, our hydrogel allows the seeded iPSCs and iPSC neurons to simply infiltrate the gel. RESULTS: IPSC-neurons grew to a depth of 500 µm into the hydrogel. Cell viability was comparable to 2D cultures over the course of three weeks, with even better neuronal survival in 3D cultures at the one-week time point. Levels of neuronal and synaptic maturation markers, namely, neural cell adhesion molecule 1 (NCAM1) and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor subunit GluR2, were strongly increased in 3D cultures. Furthermore, we identified 4-repeat (4R) tau in 3D cultures, which was not detectable in 2D cultures. CONCLUSIONS: We describe a simple, hydrogel-based method for 3D iPSC culture that can serve as a fast and drug-screening-compatible platform to identify new mechanisms and therapeutic targets for brain diseases. We further provided evidence for the increased maturation of iPSC neurons in a 3D microenvironment.

Autologous endothelialized small-caliber vascular grafts engineered from blood-derived induced pluripotent stem cells.

An ideal cell source for human therapeutic and disease modeling applications should be easily accessible and possess unlimited differentiation and expansion potential. Human induced pluripotent stem cells (hiPSCs) derived from peripheral blood mononuclear cells (PBMCs) represent a promising source given their ease of harvest and their pluripotent nature. Previous studies have demonstrated the feasibility of using PBMC-derived hiPSCs for vascular tissue engineering. However, so far, no endothelialization of hiPSC-derived tissue engineered vascular grafts (TEVGs) based on fully biodegradable polymers without xenogenic matrix components has been shown. In this study, we have generated hiPSCs from PBMCs and differentiated them into αSMA- and calponin-positive smooth muscle cells (SMCs) as well as endothelial cells (ECs) positive for CD31, vWF and eNOS. Both cell types were co-seeded on PGA-P4HB starter matrices and cultured under static or dynamic conditions to induce tissue formation in vitro. The resulting small diameter vascular grafts showed abundant amounts of extracellular matrix, containing a thin luminal layer of vWF-positive cells and a subendothelial αSMA-positive layer approximating the architecture of native vessels. Our results demonstrate the successful generation of TEVGs based on SMCs and ECs differentiated from PBMC-derived hiPSC combined with a biodegradable polymer. These results pave the way for developing autologous PBMC-derived hiPSC-based vascular constructs for therapeutic applications or disease modeling. STATEMENT OF SIGNIFICANCE: We report for the first time the possibility to employ human peripheral blood mononuclear cell (PBMC)-derived iPSCs to generate biodegradable polymer-based tissue engineered vascular grafts (TEVG), which mimic the native layered architecture of blood vessels. hiPSCs from PBMCs were differentiated into smooth muscle cells as well as endothelial cells. These cells were co-seeded on a biodegradable PGA/P4HB scaffold and cultured in a bioreactor to induce tissue formation in vitro. The resulting small diameter TEVG showed abundant amounts of extracellular matrix, containing a αSMA-positive layer in the interstitium and a thin luminal layer of vWF-positive endothelial cells approximating the architecture of native vessels. Our findings improving the generation of autologous vascular replacements using blood as an easily accessible cell source.

Oxidative stress and altered mitochondrial protein expression in the absence of amyloid-ß and tau pathology in iPSC-derived neurons from sporadic Alzheimer's disease patients.

Mitochondrial dysfunction is a prominent feature of Alzheimer's disease (AD) and increased production of reactive oxygen species (ROS) has been described in postmortem brain samples and animal models. However, these observations were made at a late stage of disease and the inability to examine an early, presymptomatic phase in human neurons impeded our understanding of cause or consequence of mitochondrial dysfunction in AD. We used human induced pluripotent stem cell-derived neuronal cells (iN cells) from sporadic AD (SAD) patients and healthy control subjects (HCS) to show aberrant mitochondrial function in patient-derived cells. We observed that neuronal cultures from some patients produced more ROS and displayed higher levels of DNA damage. Furthermore, patient-derived cells showed increased levels of oxidative phosphorylation chain complexes, whereas mitochondrial fission and fusion proteins were not affected. Surprisingly, these effects neither correlated with Aß nor phosphorylated and total tau levels. Synaptic protein levels were also unaffected in SAD iN cells. The results of this study give new insights into constitutional metabolic changes in neurons from subjects prone to develop Alzheimer's pathology. They suggest that increased ROS production may have an integral role in the development of sporadic AD prior to the appearance of amyloid and tau pathology.

Tau pathology-dependent remodelling of cerebral arteries precedes Alzheimer's disease-related microvascular cerebral amyloid angiopathy.

Alzheimer's disease (AD) is characterised by pathologic cerebrovascular remodelling. Whether this occurs already before disease onset, as may be indicated by early Braak tau-related cerebral hypoperfusion and blood-brain barrier (BBB) impairment found in previous studies, remains unknown. Therefore, we systematically quantified Braak tau stage- and cerebral amyloid angiopathy (CAA)-dependent alterations in the alpha-smooth muscle actin (α-SMA), collagen, and elastin content of leptomeningeal arterioles, small arteries, and medium-sized arteries surrounding the gyrus frontalis medialis (GFM) and hippocampus (HIPP), including the sulci, of 17 clinically and pathologically diagnosed AD subjects (Braak stage IV-VI) and 28 non-demented control subjects (Braak stage I-IV). GFM and HIPP paraffin sections were stained for general collagen and elastin with the Verhoeff-van Gieson stain; α-SMA and CAA/amyloid ß (Aß) were detected using immunohistochemistry. Significant arterial elastin degradation was observed from Braak stage III onward and correlated with Braak tau pathology (ρ = 0.909, 95% CI 0.370 to 0.990, p < 0.05). This was accompanied by an increase in neutrophil elastase expression by α-SMA-positive cells in the vessel wall. Small and medium-sized arteries exhibited significant CAA-independent α-SMA loss starting between Braak stage I and II-III, along with accumulation of phosphorylated paired helical filament (PHF) tau in the perivascular space of intraparenchymal vessels. α-SMA remained at the decreased level throughout the later Braak stages. In contrast, arterioles exhibited significant α-SMA loss only at Braak stage V and VI/in AD subjects, which was CAA-dependent/correlated with CAA burden (ρ = -0.422, 95% CI -0.557 to -0.265, p < 0.0001). Collagen content was only significantly changed in small arteries. Our data indicate that vessel wall remodelling of leptomeningeal arteries is an early-onset, Braak tau pathology-dependent process unrelated to CAA and AD, which potentially may contribute to downstream CAA-dependent microvascular pathology in AD.