intracerebral-transplantation-vivo-bioluminescence-tracking-human

Intracerebral Transplantation and <em>In Vivo</em> 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 ...

University of Zurich

Biodegradable scaffold matrixes form the basis of any in vitro tissue engineering approach by acting as a temporary matrix for cell proliferation and extracellular matrix deposition until the scaffold is replaced by neo-tissue. In this context several synthetic polymers have been investigated, however a concise systematic comparative analyses is missing. Therefore, the present study systematically compares three frequently used polymers for the in vitro engineering of extracellular matrix based on poly-glycolic acid (PGA) under static as well as dynamic conditions. Ultra-structural analysis was used to examine the polymers structure. For tissue engineering (TE) three human fibroblast cell lines were seeded on either PGA-poly-4-hydroxybutyrate (P4HB), PGA-poly-lactic acid (PLA) or PGA-poly-caprolactone (PCL) patches. These patches were analyzed after 21days of culture qualitative by histology and quantitative by determining the amount of DNA, glycosaminoglycan and hydroxyproline. We found that PGA-P4HB and PGA-PLA scaffolds enhance tissue formation significantly higher than PGA-PCL scaffolds (p<0.05). Polymer remnants were visualized by polarization microscopy. In addition, biomechanical properties of the tissue engineered patches were determined in comparison to native tissue. This study may allow future studies to specifically select certain polymer starter matrices aiming at specific tissue properties of the bioengineered constructs in vitro.

Comparative analysis of poly-glycolic acid-based hybrid polymer starter matrices for in vitro tissue engineering.

Amniotic fluid represents an abundant source of multipotent stem cells, referred as broadly multipotent given their differentiation potential and expression of pluripotency-related genes. However, the origin of this broadly multipotent cellular fraction is not fully understood. Several sources have been proposed so far, including embryonic and extraembryonic tissues. In this regard, the ovine developmental model uniquely allows for direct comparison of fetal fluid-derived cells from two separate fetal fluid cavities, the allantois and the amnion, over the entire duration of gestation. As allantoic fluid mainly collects fetal urine, cells originating from the efferent urinary tract can directly be compared with cells deriving from the extraembryonic amniotic tissues and the fetus. This study shows isolation of cells from the amniotic [ovine amniotic fluid cells (oAFCs)] and allantoic fluid [ovine allantoic fluid cells (oALCs)] in a strictly paired fashion with oAFCs and oALCs derived from the same fetus. Both cell types showed cellular phenotypes comparable to standard mesenchymal stem cells (MSCs), with trilineage differentiation potential, and expression of common ovine MSC markers. However, the expression of MSC markers per single cell was higher in oAFCs as measured by flow cytometry. oAFCs exhibited higher proliferative capacities and showed significantly higher expression of pluripotency-related genes OCT4, STAT3, NANOG, and REX1 by quantitative real-time polymerase chain reaction compared with paired oALCs. No significant decrease of pluripotency-related gene expression was noted over gestation, implying that cells with high differentiation potential may be isolated at the end of pregnancy. In conclusion, this study suggests that cells with highest stem cell characteristics may originate from the fetus itself or the amniotic fetal adnexa rather than from the efferent urinary tract or the allantoic fetal adnexa.

Amniotic Fluid Cells Show Higher Pluripotency-Related Gene Expression Than Allantoic Fluid Cells.

Human mesenchymal stromal cell (hMSC) secretomes have shown to influence the microenvironment upon injury, promoting cytoprotection, angiogenesis, and tissue repair. The angiogenic potential is of particular interest for the treatment of ischemic diseases. Interestingly, hMSC secretomes isolated from different tissue sources have shown dissimilarities with respect to their angiogenic profile. This study compares angiogenesis of hMSC secretomes from adipose tissue (hADSCs), bone marrow (hBMSCs), and umbilical cord Wharton's jelly (hWJSCs). hMSC secretomes were obtained under xenofree conditions and analyzed by liquid chromatography tandem mass spectrometry (LC/MS-MS). Biological processes related to angiogenesis were found to be enriched in the proteomic profile of hMSC secretomes. hWJSC secretomes revealed a more complete angiogenic network with higher concentrations of angiogenesis related proteins, followed by hBMSC secretomes. hADSC secretomes lacked central angiogenic proteins and expressed most detected proteins to a significantly lower level. In vivo all secretomes induced vascularization of subcutaneously implanted Matrigel plugs in mice. Differences in secretome composition were functionally analyzed with monocyte and endothelial cell (EC) in vitro co-culture experiments using vi-SNE based multidimensional flow cytometry data analysis. Functional responses between hBMSC and hWJSC secretomes were comparable, with significantly higher migration of CD14 CD16 monocytes and enhanced macrophage differentiation compared with hADSC secretomes. Both secretomes also induced a more profound pro-angiogenic phenotype of ECs. These results suggest hWJSCs secretome as the most potent hMSC source for inflammation-mediated angiogenesis induction, while the potency of hADSC secretomes was lowest. This systematic analysis may have implication on the selection of hMSCs for future clinical studies.

Proteomic analysis of human mesenchymal stromal cell secretomes: a systematic comparison of the angiogenic potential.

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.

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

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.

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

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.

Article	Year
Comparative analysis of poly-glycolic acid-based hybrid polymer starter matrices for in vitro tissue engineering. Colloids and surfaces. B, Biointerfaces\| PubMed ID: 28697435	2017
Amniotic Fluid Cells Show Higher Pluripotency-Related Gene Expression Than Allantoic Fluid Cells. Stem cells and development\| PubMed ID: 28708036	2017
Proteomic analysis of human mesenchymal stromal cell secretomes: a systematic comparison of the angiogenic potential. NPJ Regenerative medicine\| PubMed ID: 31016031	2019
Autologous endothelialized small-caliber vascular grafts engineered from blood-derived induced pluripotent stem cells. Acta biomaterialia\| PubMed ID: 31344511	2019
High Frequency of Tumor Propagating Cells in Fusion-Positive Rhabdomyosarcoma. Genes\| PubMed ID: 34573355	2021
Heterogeneous expression of ACE2 and TMPRRS2 in mesenchymal stromal cells. Journal of cellular and molecular medicine\| PubMed ID: 34821008	2022

Melanie Generali

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