Dokument: Establishment of Human Amniotic Fluid and Urine as Sources for Mesenchymal Stem Cells of Renal Origin with Versatile Regenerative Potential
| Titel: | Establishment of Human Amniotic Fluid and Urine as Sources for Mesenchymal Stem Cells of Renal Origin with Versatile Regenerative Potential | |||||||
| URL für Lesezeichen: | https://docserv.uni-duesseldorf.de/servlets/DocumentServlet?id=56126 | |||||||
| URN (NBN): | urn:nbn:de:hbz:061-20210510-074311-0 | |||||||
| Kollektion: | Dissertationen | |||||||
| Sprache: | Englisch | |||||||
| Dokumententyp: | Wissenschaftliche Abschlussarbeiten » Dissertation | |||||||
| Medientyp: | Text | |||||||
| Autor: | M.Sc Rahman, Md Shaifur [Autor] | |||||||
| Dateien: |
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| Beitragende: | Prof. Dr. Adjaye, James [Gutachter] Prof. Dr. Czekelius, Constantin [Gutachter] | |||||||
| Dewey Dezimal-Klassifikation: | 500 Naturwissenschaften und Mathematik » 570 Biowissenschaften; Biologie | |||||||
| Beschreibung: | Investigations on stem cells and cell-based therapies are rising as an alternative of conventional treatment options. Amongst all cells, multipotent stem cells have been established as most promising for clinical uses. For instance, human mesenchymal stem cells (MSCs) have already been extensively examined in clinical trials and already have found their way into the clinic to a certain extend. MSCs are immune-privileged, able to self-renew, highly proliferative, and have a broad differentiation potential. Bone marrow (BM), adipose and early trimester amniotic fluid (AF) are well-established sources of MSCs with broad regenerative potential. However, shortage of donors and invasive collection procedures- are the limitations associated with risks and pain. Since numerous Caesarean sections are done each year, we used this opportunity to collect amniotic fluid without any additional risk for the mother and the foetus. Subsequently we isolated AF cells, cultured and characterized them. It could be shown that these cells fulfil the criteria for MSCs set by the International Society for Cellular Therapy (ISCT). Characteristically AF-MSCs able to adhere on plastic, possess fibroblastoid morphology, express cell surface markers CD73, CD90, and CD105, they are positive for Vimentin, CD133 and negative for E-Cadherin. In vitro AF-MSCs differentiate into bone, fat and cartilage cells. Furthermore, transcriptome analyses revealed similarities with foetal BM-derived MSCs. Thus, we confirmed third trimester AF as a non-invasive source of MSCs with regenerative potential for clinical applications.
Before using these cells in clinic, it is necessary to solve the controversial issues related to the exact origin of AF-MSCs; which was the focus in the second part of this work. We know that AF is mostly composed of foetal urine; logically we postulated that AF cells were exfoliated from foetal renal system and deposited in amniotic sac. Irrespective of media used, subpopulations of AF-MSCs were observed morphologically alike renal progenitors and expressed pluripotency-associated markers such as SSEA4, TRA-1-60, TRA-1-81 and c-KIT. Importantly, AF-MSCs express the key renal stem cell proteins SIX2 and CITED1, together with other renal markers when compared to commercially available kidney biopsy-derived renal cells (HREpCs). We also observed AF-MSCs have renal cell transporter functionality. Transcriptome analyses of AF-MSCs revealed more similarities with human kidney cells than with foetal MSCs, and the GO terms identified biological processes associated with kidney morphogenesis. Gene expression and semi-qPCR analysis indicated an increasing expression level and number of renal genes with gestational time, which reflects a synergistic relationship between renal gene expression and foetal nephrogenesis with pregnancy periods. To understand how self-renewal and gene regulation maintenance of these cells is controlled in vitro, we activated WNT signaling using the GSK3ß inhibitor (CHIR99201). Upon this disruption of self-renewal, AF-MSCs were elongated in morphology and had decreased c-KIT, SIX2 and CD133 expression. Taken above context together, third-trimester amniotic fluid could serve as a novel source of renal progenitor cells. Increasing number of patients with kidney-associated dysfunctions urge for stem cell-based therapeutic options. Due to insufficient access to renal progenitor cells, finding alternatives sources of these cells is very important for not only cell-based therapeutic purpose but also for in vitro renal disease modelling and drug screening. Based on this need, the purpose of the third part of this thesis were investigations on adult human urine as a possible solution. It could be shown that adult human urine represents a noninvasive, repetitive, inexpensive and robust source of renal stem cells originating from the kidney. Like full term AF-MSCs, urine-derived renal progenitor cells (UdRPCs) are multipotent MSCs and express a subset of pluripotency-related proteins. Remarkably, UdRPCs express renal stem cell markers such as SIX2, CITED1, WT1 and show transporter functionality. Unmethylated CpG islands within the vicinity of SIX2 5′- regulatory region further denote their progenitor status. Transcriptome based cluster analysis revealed a clear-cut separation between undifferentiated UdRPCs and differentiated HREpCs. Differentiation of UdRPCs using CHIR99021 showed upregulation of WNT- associated genes- AXIN2, JUN and NKD1. In the self-renewal disrupted UdRPCs, a suppression of FGF2, FGF7 and over expression of BMP7, BMP4 were identified at transcriptome level. To validate, downregulation of SIX2 were detected in CHIR99021 treated as well as SU5402 (FGFR inhibitor) treated cells. Further analyses indicated that self-renewal of UdRPCs in vitro is sustained through FGF2-driven TGFβ- SMAD2/3 pathway. These SIX2+UdRPCs with known CYP2D6 status represents as a potential stem cell types which might be applicable for modelling renal-associated diseases, nephrotoxicity studies, drug screening and in future cell-replacement therapy. Like full-term AF-MSCs, UdRPCs’ are not pluripotent but express a subset of pluripotency-associated proteins which could facilitated swift converting of these cells into induced pluripotent stem cells (iPSCs). We generated stable AF-iPSCs and UdRPCs-iPSCs using nucleofection of episomal-based plasmids containing OCT4, SOX2, c-MYC, LIN28, and KLF4 without perturbations of TGFβ, MEK and GSK3β pathways. AF-iPSCs and UdRPCs-iPSCs can be used for renal disease modelling and 3D kidney organoid development. In addition to the research on AF and urine-derived stem cells, we investigated the potential of iPSCs to generate cell types which are valuable for regenerative therapies. Primary MSCs obtained from adult donors are largely associated with several disadvantages such as ageing, limited autologous cell number and limited replicative potential in culture. To overcome this, the differentiated iPSCs into MSCs (iMSCs) could serve as an alternative of native MSCs. Induced MSCs possess all the criteria of bon-fide MSCs and overcome the ageing signature as it was derived through iPSCs. Upon transplantation of iMSCs together with the scaffold material CPG (calcium phosphate granule) into a proximal tibia defect of pigs a beneficial regenerative effect was identified which was similar to the treatment with autologous BMC (Bone marrow concentrate) together with CPG. There is increasing interest on the fabrication of 3D scaffolds from naturally available osteo-conductive materials to mimic the structural support for bone defect improvement. Therefore, we engineered biocompatible porous scaffolds consist of hydroxyapatite, collagen and chitosan (Ha-Col1-Cs); and were found to improve defected maxillofacial mandible bone of rat upon grafting. For optimal and more effective bone healing, a combination of AF-MSCs or uMSCs or iMSCs and Ha-Col1-Cs scaffolds need to be experimented for the repair of large bone defects. As a further use of naturally available biomaterial, gels formulated from amnion, collagen and Aloe vera in different combinations and tested for their wound healing potency using an induced burn wound rat model. Application of the different gels could show a significant improved healing of the wound. Potentially these gels could be combined with stem cell derived skin cells to increase treatment effects. To study this, we were able to differentiate the full-term AF-MSCs into keratinocyte like cells (AF-KC) expressing epidermal markers- K5, K14 and K10. Taken all together this study shows a broad range of stem cells sources and versatile application thereof. The findings of this study can contribute to drive forward regenerative medicine in various fields. | |||||||
| Lizenz: |  Urheberrechtsschutz | |||||||
| Fachbereich / Einrichtung: | Mathematisch- Naturwissenschaftliche Fakultät | |||||||
| Dokument erstellt am: | 10.05.2021 | |||||||
| Dateien geändert am: | 10.05.2021 | |||||||
| Promotionsantrag am: | 28.11.2020 | |||||||
| Datum der Promotion: | 24.03.2021 |
