Reprogramming Of Mesenchymal Stem Cells Research Articles

The role of TNF-α in the fate regulation and functional reprogramming of mesenchymal stem cells in an inflammatory microenvironment.

Mesenchymal stem cells (MSCs) are pluripotent stem cells with multidirectional differentiation potential and strong immunomodulatory capacity. MSCs have been widely used in the treatment of injured, inflammatory, and immune-related diseases. Resting MSCs lack differentiation and immunomodulatory ability. Instead, they rely on microenvironmental factors to: 1) stimulate and regulate their expression of specific cell growth factors, chemokines, immunomodulatory factors, or receptors; or 2) direct their differentiation into specific tissue cells, which ultimately perform tissue regeneration and repair and immunomodulatory functions. Tumor necrosis factor (TNF)-α is central to the creation of an inflammatory microenvironment. TNF-α regulates the fate and functional reprogramming of MSCs, either alone or in combination with a variety of other inflammatory factors. TNF-α can exert opposing effects on MSCs, from inducing MSC apoptosis to enhancing their anti-tumor capacity. In addition, the immunomodulation and osteogenic differentiation capacities of MSCs, as well as their exosome or microvesicle components vary significantly with TNF-α stimulating concentration, time of administration, or its use in combination with or without other factors. Therefore, this review discusses the impact of TNF-α on the fate and functional reprogramming of MSCs in the inflammatory microenvironment, to provide new directions for improving the immunomodulatory and tissue repair functions of MSCs and enhance their therapeutic potential.

Abstract IA009: Oncogenic transcription factors drive highly tumor-specific LincRNA

Abstract Many cancers are characterized by gene fusions encoding oncogenic chimeric transcription factors (TFs) such as EWS::FLI1 in Ewing sarcoma (EwS). EWS::FLI1 induces a complete reprogramming of mesenchymal stem cells, the putative cell of origin of Ewing sarcoma, through binding and subsequent generation of thousands of neoenhancers at GGAA microsatellite repeats. We show that EWS::FLI1 induces the robust expression of a specific set of novel spliced and polyadenylated transcripts within otherwise transcriptionally silent regions of the genome. These neo-genes (NGs) are virtually undetectable in large collections of normal tissues or non-EwS tumors and can be silenced by CRISPR interference at regulatory EWS::FLI1-bound microsatellites. Ribosome profiling and proteomics further show that some NGs are translated into highly EwS-specific peptides. More generally, hundreds of NGs can be detected in diverse cancers characterized by chimeric TFs. Altogether, we identify the transcription, processing, and translation of novel, specific, highly expressed multi-exonic transcripts from otherwise silent regions of the genome as a new activity of aberrant TFs in cancer. These neotranscripts, which can be classified as LincRNAs, or neopeptides may have specific functions in the tumor cells. The latter may also constitute ideal neoepitopes as they are tumor-specific, clonally expressed in sarcoma cells, induced by the driver genetic alterations and public for a given sarcoma. Citation Format: Olivier Delattre, Julien Vibert, Olivier Saulnier, Joshua Waterfall. Oncogenic transcription factors drive highly tumor-specific LincRNA [abstract]. In: Proceedings of the AACR Special Conference: Sarcomas; 2022 May 9-12; Montreal, QC, Canada. Philadelphia (PA): AACR; Clin Cancer Res 2022;28(18_Suppl):Abstract nr IA009.

Xeno- and transgene-free reprogramming of mesenchymal stem cells toward the cells expressing neural markers using exosome treatments.

Neural stem cells (NSCs), capable of self-renew and differentiate into neural cells, hold promise for use in studies and treatments for neurological diseases. However, current approaches to obtain NSCs from a live brain are risky and invasive, since NSCs reside in the subventricular zone and the in the hippocampus dentate gyrus. Alternatively, mesenchymal stem cells (MSCs) could be a more available cell source due to their abundance in tissues and easier to access. However, MSCs are committed to producing mesenchymal tissue and are not capable of spontaneously differentiating into neural cells. Thus, the process of reprogramming of MSCs into neural cells to use in clinical and scientific settings has significantly impacted the advancement of regenerative medicine. Previously, our laboratory reported trans-differentiation of MSCs to neural cells through the induced pluripotent stem (iPS) cells state, which was produced by overexpression of the embryonic stem cell gene NANOG. In the current study, we demonstrate that treatment with exosomes derived from NSCs makes MSCs capable of expressing neural cell markers bypassing the generation of iPS cells. An epigenetic modifier, decitabine (5-aza-2'-deoxycytidine), enhanced the process. This novel Xeno and transgene-free trans-differentiation technology eliminates the issues associated with iPS cells, such as tumorigenesis. Thus, it may accelerate the development of neurodegenerative therapies and in vitro neurological disorder models for personalized medicine.

Abstract B55: Epigenetic reprogramming of mesenchymal stem cells by ovarian carcinoma to facilitate metastasis

Abstract Ovarian cancer, the most deadly gynecologic cancer, resides within a complex tumor microenvironment that supports cancer growth, survival, and spread. Carcinoma-associated mesenchymal stem cells (CA-MSCs) are stromal stem cells within the ovarian TME that strongly support tumor growth promoting cancer initiation, increasing the cancer stem cell pool and enhancing chemotherapy resistance. We recently demonstrated that ovarian cancer stimulation converts normal tissue-derived MSCs into CA-MSCs. The goal of this work is to investigate the mechanism of cancer-mediated CA-MSC formation. We hypothesize that ovarian cancer stimulation induces the epigenetic reprogramming of MSCs creating CA-MSCs. EPIC array DNA methylation analysis, histone mass spectrometry, and Assay for Transposase-Accessible Chromatin using sequencing (ATACseq) were performed on CA-MSCs derived from ovarian cancer patient samples and normal MSCs derived from benign fallopian tube and omentum. We demonstrate that CA-MSCs have large-scale DNA methylation differences vs. normal tissue MSCs. This DNA methylation pattern is distinct from cancer cells; however, the CA-MSC methylation pattern is represented in the DNA methylation pattern of bulk tumor from clear-cell, endometrioid, and high-grade serous cancers, indicating the importance of stromal contribution to bulk tumor analysis. DNA methylation changes were acquired during the conversion of a normal MSC into a CA-MSC and retained after CA-MSC adipocyte and fibroblast differentiation. Histone mass spectrometry and ATACseq demonstrated enrichment in repressive histone marks and overall decreased ATACseq peaks consistent with altered chromosome accessibility. Integration of DNA methylation, ATACseq, and RNAseq data demonstrates strong correlation between epigenetic changes and transcriptomic changes. Interestingly, cell:cell adhesion pathways were consistently upregulated consistent with a partial mesenchymal-to-epithelial transition (MET). This partial MET engenders CA-MSCs with enhanced tumor cell-binding capacity. Direct tumor cell:CA-MSC binding enhanced ovarian cancer migration, extravasation, and nonadherent survival in vitro and increased ovarian cancer metastasis in vivo. This partial MET may be mediated via WT1, a mediator of MET during development, as knockdown of WT1 prevented the formation of a CA-MSCs while overexpression of WT1 enhances the formation of a CA-MSC. Collectively, we demonstrate ovarian cancer stimulates the epigenetic reprogramming of normal MSCs into CA-MSCs, thus enhancing direct tumor cell binding, which facilitates ovarian cancer metastasis. Further investigation is needed to identify the molecular drivers of this reprogramming to allow the disruption of the protumorigenic TME, which may represent a powerful approach to the treatment of ovarian cancer. Citation Format: Leonard Frisbie, HuiHui Fan, Thomas Pisanic, Hui Shen, Lan Coffman. Epigenetic reprogramming of mesenchymal stem cells by ovarian carcinoma to facilitate metastasis [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research; 2019 Sep 13-16, 2019; Atlanta, GA. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(13_Suppl):Abstract nr B55.

Development of Hepatocyte-like Cell Derived from Human Induced Pluripotent Stem cell as a Host for Clinically Isolated Hepatitis C Virus.

This unit describes protocols to develop hepatocyte‐like cells (HLCs) starting from mesenchymal stem cells (MSCs) as a natural host for hepatitis C virus (HCV). These include the preparation of MSCs from bone marrow, the reprogramming of MSCs into induced pluripotent stem cells (iPSCs), and the differentiation of iPSCs into HLCs. This unit also incorporates the characterization of the resulting cells at each stage. Another section entails the preparations of HCV. The sources of HCV are either the clinically isolated HCV (HCVser) and the conventional JFH‐1 genotype. The last section is the infection protocol coupled with the measurement of viral titer. © 2017 by John Wiley & Sons, Inc.

Metabolic Reprogramming of Mesenchymal Stem Cells upon Co-Cultivation with Primary CLL Cells Determined By Mass Spectrometry-Based Proteomics

Chronic lyhocytic leukemia displays a strong dependency on tumor-stroma interactions. CLL cells were found to rapidly die when cultivated in vitro, whereas co-cultivation with mesenchymal stromal cells promotes cell proliferation and ensures leukemia cell survival. It is therefore of great interest to obtain a thourough picture of the exact molecular players involved in this critical interplay. While a number of protagonists are already well described, there are still unresoved issues concerning the effect of stromal cells on the metabolism of CLL cells.In this study we co-cultivated for one week primary CLL cells obtained from peripheral blood with mesenchymal cells (hMSC). Subsequently, hMSC were lysed and fractionated to obtain cytoplamic, nuclear and secreted proteins. To allow comparative analyses, B-cells from healthy individuals and hMSC were co-cultivated.Mass spectrometry-based proteomic analysis resulted in the identification and relative quantification of more than 3700 proteins. Comparative analyses between co-cultures with CLL and healthy B-cells showed that already at a ratio of 0.25% CLL cells could induce a significant proteomic alteration in hMSC. Furthermore, hMSC were found to undergo a metabolic change upon co-cultivation with CLL cells, an effect which was most pronounced in the secreted protein fraction. Annotation analyses according to gene ontology revealed that proteins related to "generation of precursor metaboites" were significantly up-regulated in hMSC upon co-culture with CLL cells. This indicated that these stromal cells "feed" the leukemia cells by producing and secreting high energy metabolite, satisfying the elevated metabolic demands of the CLL cells.Revealing the metabolic interactions between CLL cells and surrounding stromal cells by mass spetrometry-based methods should contribute to the development of innovative treatment strategies. DisclosuresNo relevant conflicts of interest to declare.

Tumor Specific Recruitment and Reprogramming of Mesenchymal Stem Cells in Tumorigenesis.

Non-neoplastic stromal cells harvested from patient tumors were identified as tumor-derived mesenchymal stem cells (MSCs) by their multipotential capacity to differentiate into adipocytes, osteoblasts, and chondrocytes and by the expression of MSC specific cell surface markers. These procedures yielded also epithelial cancer cells and their counterpart MSC from gastric carcinoma (GSC1) and lung carcinoma (LC2). While the LC2 cancer cell growth is independent of their LC-MSC, the GSC1 cancer cell growth is critically dependent on the presence of their counterpart GSC-MSC or their conditioned medium (CM). The fact that none of the various other tumor-derived MSCs was able to restore the specific effect of GSC-MSC on GSC1 cancer cell growth suggests specificity of tumor-derived MSC, which are specifically recruited and "educated"/reprogrammed by the cancer cells to support tumor growth. Using cytokine array analysis, we were able to demonstrate that GSC1 cell growth is mediated through hepatocyte growth factor (HGF)/c-MET signaling pathway which is activated exclusively by HGF secreted from GSC-MSC. An innovative approach demonstrates GSC1-mediated specific tropism of "naïve" MSC from the adjacent tissue in a tumor specific manner to support tumor progression. The results suggest that specific tumor tropic "naïve" MSC are reprogrammed in a tumor-specific manner to support gastric tumor progression. Understanding the mechanisms involved in the interactions of the tumor cancer cells and tumor-derived MSC will constitute the basis for developing multimodal anticancer therapeutic strategies that will also take into account the specific tumor tropism properties of MSC and their reprogramming.

Abstract 49: Targeting Endothelial Cells with HO-1 Attenuated Vascular and Adipocyte Dysfunction in Mice Fed High Fat Diet

Introduction: Obesity or high fat diet (HF) are risk factors for the development of hypertension. We examined the hypothesis that targeting the vascular system with human heme oxygenase -1 (HO-1) may attenuate both vascular and adipocyte dysfunction in vivo and in vitro. Methods: Lentivirus (Lenti) construct expressing human HO-1 under the control of endothelium specific promoters VE-cadherin (VECAD-HO-1) and VECAD −GFP (control) were used to treat mice, using a bolus injection into the renal artery, and kept on a high fat diet for 26 weeks. Human HO-1 gene expression was sustained for 9 months. For in vitro studies, human EC were cultured with Lenti- VECAD-HO-1 and added to Lenti-VECAD-GFP or control. The conditioned media (CM) from ECs was harvested and 10% CM was added to adipogenic media to measure paracrine effect on adipogenesis in human Mesenchymal Stem Cells (MSCs) derived adipocytes. Signaling molecules were measured by western blot. Results: Lentiviral transduction with VECAD-HO-1 construct attenuated the increase in blood pressure (from 149.9 ± 2.4 to 118 ± 2.0 mmHg, p<0.01) and prevented body weight gain by 39% (p<0.05) in obese mice and increased plasma adiponectin (from 2.9 ± 0.2 to 6.5 ± 0.1 μg/ml, p<0.05). CM derived from EC lenti HO-1 decreased adipogenesis compared to control (from 17.0 ± 0.2 to 10.2 ± 0.1, p<0.05) and resulted in an increase of β-catenin and Wnt but a decrease in PPARγ (p<0.05). These beneficial effects were reversed by treating the cell with SnMP, an inhibitor of HO activity (p<0.01). EC-HO-1 increased angiopoietin-1 (ANG-1), vascular endothelial growth factor A (VEGF), and platelet derived growth factor (PDGF)-AA,−BB (p<0.05). The addition of ANG-1 to adipogenic media resulted in the inhibition of adipogenesis (8.1 ± 0.1, p<0.01). Conclusion: Targeting HO-1 to ECs resulted in the attenuation of blood pressure and the prevention of body weight gain resulting in increased ANG-1, PDGF, and VGEF levels and the reprogramming of MSCs derived adipocytes to produce healthy and smaller adipocytes with the release of adiponectin.

Reprogramming of mesenchymal stem cells derived from iPSCs seeded on biofunctionalized calcium phosphate scaffold for bone engineering

Human induced pluripotent stem cell-derived mesenchymal stem cells (iPSC-MSCs) are a promising choice of patient-specific stem cells with superior capability of cell expansion. There has been no report on bone morphogenic protein 2 (BMP2) gene modification of iPSC-MSCs for bone tissue engineering. The objectives of this study were to: (1) genetically modify iPSC-MSCs for BMP2 delivery; and (2) to seed BMP2 gene-modified iPSC-MSCs on calcium phosphate cement (CPC) immobilized with RGD for bone tissue engineering. iPSC-MSCs were infected with green fluorescence protein (GFP-iPSC-MSCs), or BMP2 lentivirus (BMP2-iPSC-MSCs). High levels of GFP expression were detected and more than 68% of GFP-iPSC-MSCs were GFP positive. BMP2-iPSC-MSCs expressed higher BMP2 levels than iPSC-MSCs in quantitative RT-PCR and ELISA assays (p < 0.05). BMP2-iPSC-MSCs did not compromise growth kinetics and cell cycle stages compared to iPSC-MSCs. After 14 d in osteogenic medium, ALP activity of BMP2-iPSC-MSCs was 1.8 times that of iPSC-MSCs (p < 0.05), indicating that BMP2 gene transduction of iPSC-MSCs enhanced osteogenic differentiation. BMP2-iPSC-MSCs were seeded on CPC scaffold biofunctionalized with RGD (RGD-CPC). BMP2-iPSC-MSCs attached well on RGD-CPC. At 14 d, COL1A1 expression of BMP2-iPSC-MSCs was 1.9 times that of iPSC-MSCs. OC expression of BMP2-iPSC-MSCs was 2.3 times that of iPSC-MSCs. Bone matrix mineralization by BMP2-iPSC-MSCs was 1.8 times that of iPSC-MSCs at 21 d. In conclusion, iPSC-MSCs seeded on CPC were suitable for bone tissue engineering. BMP2 gene-modified iPSC-MSCs on RGD-CPC underwent osteogenic differentiation, and the overexpression of BMP2 in iPSC-MSCs enhanced differentiation and bone mineral production on RGD-CPC. BMP2-iPSC-MSC seeding on RGD-CPC scaffold is promising to enhance bone regeneration efficacy.

Derivation of islet‐like cells from mesenchymal stem cells using PDX1‐transducing lentiviruses

Pancreatic duodenum homeobox protein-1 (PDX1) is a master regulatory gene in pancreatic development. Reprogramming of mesenchymal stem cells (MSCs) is a promising tool for producing insulin-producing cells. In this study, lentivirus harboring PDX1 (LV-PDX1) has been used for persistence gene expression in MSCs. The objective of this study was to evaluate the potential of lentivirus to introduce the PDX1 gene into MSCs to produce insulin-secreting cells and apply it for treatment of hyperglycemia in diabetic rats. MSCs were isolated from rat bone marrow, characterized, and transduced by LV-PDX1. Significant expressions of PDX1, neurogenin3, glucagon, glucose transporter2 (Glut2), and insulin were detected by quantitative reverse transcription-polymerase chain reaction (P < 0.05). PDX1 and insulin were detected at the protein level by immunofluorescence analysis. PDX1 could trigger a gene expression cascade that involved pancreatic endocrine differentiation and also revealed the glucose sensing ability by expressing Glut2 in high-glucose medium. The insulin secretion of MSCs(PDX1+) in the high-glucose medium was 1.75-fold higher than that secreted in the low-glucose medium (P < 0.05). MSCs(PDX1+) implanted into diabetic rats could decrease the blood glucose level from 485 mg/dL to the normal level in 3 days. This study showed MSCs(PDX1+) have the potential to be used as a viable resource in cell-based gene therapy of type 1 diabetes.

Reprogramming of mesenchymal stem cells by the synovial sarcoma-associated oncogene SYT-SSX2.

Cell identity is determined by its gene expression programs. The ability of a cell to change its identity and produce cell types outside its lineage is achieved by the activity of transcription controllers capable of reprogramming differentiation gene networks. The synovial sarcoma (SS)-associated protein, SYT-SSX2, reprograms myogenic progenitors and human bone marrow-derived mesenchymal stem cells (BMMSCs) by dictating their commitment to a pro-neural lineage. It fulfills this function by directly targeting an extensive array of neural-specific genes as well as genes of developmental pathway mediators. Concomitantly, the ability of both myoblasts and BMMSCs to differentiate into their normal myogenic and adipogenic lineages was compromised. SS is believed to arise in mesenchymal stem cells where formation of the t(X/18) translocation product, SYT-SSX, constitutes the primary event in the cancer. SYT-SSX is therefore believed to initiate tumorigenesis in its target stem cell. The data presented here allow a glimpse at the initial events that likely occur when SYT-SSX2 is first expressed, and its dominant function in subverting the nuclear program of the stem cell, leading to its aberrant differentiation, as a first step toward transformation. In addition, we identified the fibroblast growth factor receptor gene, Fgfr2, as one occupied and upregulated by SYT-SSX2. Knockdown of FGFR2 in both BMMSCs and SS cells abrogated their growth and attenuated their neural phenotype. These results support the notion that the SYT-SSX2 nuclear function and differentiation effects are conserved throughout sarcoma development and are required for its maintenance beyond the initial phase. They also provide the stem cell regulator, FGFR2, as a promising candidate target for future SS therapy.