Derivation and characterization of integration-free iPSC line ISRM-UM51 derived from SIX2-positive renal cells isolated from urine of an African male expressing the CYP2D6 *4/*17 variant which confers intermediate drug metabolizing activity

SIX2-positive renal cells isolated from urine from a 51 year old male of African origin bearing the CYP2D6 *4/*17 variant were reprogrammed by nucleofection of a combination of two episomal-based plasmids omitting pathway (TGFβ, MEK and GSK3β) inhibition. The induced pluripotent stem cells (iPSCs) were characterized by immunocytochemistry, embryoid body formation, DNA-fingerprinting and karyotype analysis. Comparative transcriptome analyses with human embryonic stem cell lines H1 and H9 revealed a Pearson correlation of 0.9243 and 0.9619 respectively

Fabrication of biocompatible porous scaffolds based on hydroxyapatite/collagen/chitosan composite for restoration of defected maxillofacial mandible bone

Fabrication of scaffolds from biomaterials for restoration of defected mandible bone has attained increased attention due to limited accessibility of natural bone for grafting. Hydroxyapatite (Ha), collagen type 1 (Col1) and chitosan (Cs) are widely used biomaterials which could be fabricated as a scaffold to overcome the paucity of bone substitutes. Here, rabbit Col1, shrimp Cs and bovine Ha were extracted and characterized with respect to physicochemical properties. Following the biocompatibility, degradability and cytotoxicity tests for Ha, Col1 and Cs a hydroxyapatite/collagen/chitosan (Ha·Col1·Cs) scaffold was fabricated using thermally induced phase separation technique. This scaffold was cross-linked with (1) either glutaraldehyde (GTA), (2) de-hydrothermal treatment (DTH), (3) irradiation (IR) and (4) 2-hydroxyethyl methacrylate (HEMA), resulting in four independent types (Ha·Col1·Cs-GTA, Ha·Col1·Cs-IR, Ha·Col1·Cs-DTH and Ha·Col1·Cs-HEMA). The developed composite scaffolds were porous with 3D interconnected fiber microstructure. However, Ha·Col1·Cs-IR and Ha·Col1·Cs-GTA showed better hydrophilicity and biodegradability. All four scaffolds showed desirable blood biocompatibility without cytotoxicity for brine shrimp. In vitro studies in the presence of human amniotic fluid-derived mesenchymal stem cells revealed that Ha·Col1·Cs-IR and Ha·Col1·Cs-DHT scaffolds were non-cytotoxic and compatible for cell attachment, growth and mineralization. Further, grafting of Ha·Col1·Cs-IR and Ha·Col1·Cs-DHT was performed in a surgically created non-load-bearing rabbit maxillofacial mandible defect model. Histological and radiological observations indicated the restoration of defected bone. Ha·Col1·Cs-IR and Ha·Col1·Cs-DHT could be used as an alternative treatment in bone defects and may contribute to further development of scaffolds for bone tissue engineering.</p

Human iPSC-derived MSCs (iMSCs) from aged individuals acquire a rejuvenation signature

Background: Primary mesenchymal stem cells (MSCs) are fraught with aging-related shortfalls. Human-induced pluripotent stem cell (iPSC)-derived MSCs (iMSCs) have been shown to be a useful clinically relevant source of MSCs that circumvent these aging-associated drawbacks. To date, the extent of the retention of aging-hallmarks in iMSCs differentiated from iPSCs derived from elderly donors remains unclear. Methods: Fetal femur-derived MSCs (fMSCs) and adult bone marrow MSCs (aMSCs) were isolated, corresponding iPSCs were generated, and iMSCs were differentiated from fMSC-iPSCs, from aMSC-iPSCs, and from human embryonic stem cells (ESCs) H1. In addition, typical MSC characterization such as cell surface marker expression, differentiation capacity, secretome profile, and trancriptome analysis were conducted for the three distinct iMSC preparations - fMSC-iMSCs, aMSC-iMSCs, and ESC-iMSCs. To verify these results, previously published data sets were used, and also, additional aMSCs and iMSCs were analyzed. Results: fMSCs and aMSCs both express the typical MSC cell surface markers and can be differentiated into osteogenic, adipogenic, and chondrogenic lineages in vitro. However, the transcriptome analysis revealed overlapping and distinct gene expression patterns and showed that fMSCs express more genes in common with ESCs than with aMSCs. fMSC-iMSCs, aMSC-iMSCs, and ESC-iMSCs met the criteria set out for MSCs. Dendrogram analyses confirmed that the transcriptomes of all iMSCs clustered together with the parental MSCs and separated from the MSC-iPSCs and ESCs. iMSCs irrespective of donor age and cell type acquired a rejuvenation-associated gene signature, specifically, the expression of INHBE, DNMT3B, POU5F1P1, CDKN1C, and GCNT2 which are also expressed in pluripotent stem cells (iPSCs and ESC) but not in the parental aMSCs. iMSCs expressed more genes in common with fMSCs than with aMSCs. Independent real-time PCR comparing aMSCs, fMSCs, and iMSCs confirmed the differential expression of the rejuvenation (COX7A, EZA2, and TMEM119) and aging (CXADR and IGSF3) signatures. Importantly, in terms of regenerative medicine, iMSCs acquired a secretome (e.g., angiogenin, DKK-1, IL-8, PDGF-AA, osteopontin, SERPINE1, and VEGF) similar to that of fMSCs and aMSCs, thus highlighting their ability to act via paracrine signaling. Conclusions: iMSCs irrespective of donor age and cell source acquire a rejuvenation gene signature. The iMSC concept could allow circumventing the drawbacks associated with the use of adult MSCs und thus provide a promising tool for use in various clinical settings in the future.</p

Fabrication of biocompatible porous scaffolds based on hydroxyapatite/collagen/chitosan composite for restoration of defected maxillofacial mandible bone

Fabrication of scaffolds from biomaterials for restoration of defected mandible bone has attained increased attention due to limited accessibility of natural bone for grafting. Hydroxyapatite (Ha), collagen type 1 (Col1) and chitosan (Cs) are widely used biomaterials which could be fabricated as a scaffold to overcome the paucity of bone substitutes. Here, rabbit Col1, shrimp Cs and bovine Ha were extracted and characterized with respect to physicochemical properties. Following the biocompatibility, degradability and cytotoxicity tests for Ha, Col1 and Cs a hydroxyapatite/collagen/chitosan (Haandmiddot;Col1andmiddot;Cs) scaffold was fabricated using thermally induced phase separation technique. This scaffold was cross-linked with (1) either glutaraldehyde (GTA), (2) de-hydrothermal treatment (DTH), (3) irradiation (IR) and (4) 2-hydroxyethyl methacrylate (HEMA), resulting in four independent types (Haandmiddot;Col1andmiddot;Cs-GTA, Haandmiddot;Col1andmiddot;Cs-IR, Haandmiddot;Col1andmiddot;Cs-DTH and Haandmiddot;Col1andmiddot;Cs-HEMA). The developed composite scaffolds were porous with 3D interconnected fiber microstructure. However, Haandmiddot;Col1andmiddot;Cs-IR and Haandmiddot;Col1andmiddot;Cs-GTA showed better hydrophilicity and biodegradability. All four scaffolds showed desirable blood biocompatibility without cytotoxicity for brine shrimp. In vitro studies in the presence of human amniotic fluid-derived mesenchymal stem cells revealed that Haandmiddot;Col1andmiddot;Cs-IR and Haandmiddot;Col1andmiddot;Cs-DHT scaffolds were non-cytotoxic and compatible for cell attachment, growth and mineralization. Further, grafting of Haandmiddot;Col1andmiddot;Cs-IR and Haandmiddot;Col1andmiddot;Cs-DHT was performed in a surgically created non-load-bearing rabbit maxillofacial mandible defect model. Histological and radiological observations indicated the restoration of defected bone. Haandmiddot;Col1andmiddot;Cs-IR and Haandmiddot;Col1andmiddot;Cs-DHT could be used as an alternative treatment in bone defects and may contribute to further development of scaffolds for bone tissue engineering.</p

Generation of two Alpha-I antitrypsin deficiency patient-derived induced pluripotent stem cell lines ISRM-AATD-iPSC-1 (HHUUKDi011-A) and ISRM-AATD-iPSC-2 (HHUUKDi012-A)

SIX2-positive urine derived renal progenitor cells were isolated from a male and female alpha1-antitrypsin deficiency (AATD) patients both harboring the homozygous PiZZ genotype. The cells were reprogrammed to generate two integration-free induced pluripotent stem cell (iPSC) lines by transfecting episomal-based plasmids expressing OCT4, SOX2, NANOG, c-MYC, KLF4 and LIN28. Pluripotency was confirmed by immunocytochemistry for associated markers and embryoid body-based differentiation into the three germ layers. The iPSC lines carried the parental PiZZ genotype. Comparative transcriptome analyses with human embryonic stem cell line H9 revealed a Pearson correlation of 0.945 for ISRM-AATD-iPSC-1 and 0.939 for ISRM-AATD-iPSC-2 respectively

The presence of human mesenchymal stem cells of renal origin in amniotic fluid increases with gestational time

Abstract Background Established therapies for managing kidney dysfunction such as kidney dialysis and transplantation are limited due to the shortage of compatible donated organs and high costs. Stem cell-based therapies are currently under investigation as an alternative treatment option. As amniotic fluid is composed of fetal urine harboring mesenchymal stem cells (AF-MSCs), we hypothesized that third-trimester amniotic fluid could be a novel source of renal progenitor and differentiated cells. Methods Human third-trimester amniotic fluid cells (AFCs) were isolated and cultured in distinct media. These cells were characterized as renal progenitor cells with respect to cell morphology, cell surface marker expression, transcriptome and differentiation into chondrocytes, osteoblasts and adipocytes. To test for renal function, a comparative albumin endocytosis assay was performed using AF-MSCs and commercially available renal cells derived from kidney biopsies. Comparative transcriptome analyses of first, second and third trimester-derived AF-MSCs were conducted to monitor expression of renal-related genes. Results Regardless of the media used, AFCs showed expression of pluripotency-associated markers such as SSEA4, TRA-1-60, TRA-1-81 and C-Kit. They also express the mesenchymal marker Vimentin. Immunophenotyping confirmed that third-trimester AFCs are bona fide MSCs. AF-MSCs expressed the master renal progenitor markers SIX2 and CITED1, in addition to typical renal proteins such as PODXL, LHX1, BRN1 and PAX8. Albumin endocytosis assays demonstrated the functionality of AF-MSCs as renal cells. Additionally, upregulated expression of BMP7 and downregulation of WT1, CD133, SIX2 and C-Kit were observed upon activation of WNT signaling by treatment with the GSK-3 inhibitor CHIR99201. Transcriptome analysis and semiquantitative PCR revealed increasing expression levels of renal-specific genes (e.g., SALL1, HNF4B, SIX2) with gestational time. Moreover, AF-MSCs shared more genes with human kidney cells than with native MSCs and gene ontology terms revealed involvement of biological processes associated with kidney morphogenesis. Conclusions Third-trimester amniotic fluid contains AF-MSCs of renal origin and this novel source of kidney progenitors may have enormous future potentials for disease modeling, renal repair and drug screening

Isolation and Molecular Characterization of Amniotic Fluid-Derived Mesenchymal Stem Cells Obtained from Caesarean Sections

Human amniotic fluid cells are immune-privileged with low immunogenicity and anti-inflammatory properties. They are able to self-renew, are highly proliferative, and have a broad differentiation potential, making them amenable for cell-based therapies. Amniotic fluid (AF) is routinely obtained via amniocentesis and contains heterogeneous populations of foetal-derived progenitor cells including mesenchymal stem cells (MSCs). In this study, we isolated human MSCs from AF (AF-MSCs) obtained during Caesarean sections (C-sections) and characterized them. These AF-MSCs showed typical MSC characteristics such as morphology, in vitro differentiation potential, surface marker expression, and secreted factors. Besides vimentin and the stem cell marker CD133, subpopulations of AF-MSCs expressed pluripotency-associated markers such as SSEA4, c-Kit, TRA-1-60, and TRA-1-81. The secretome and related gene ontology (GO) terms underline their immune modulatory properties. Furthermore, transcriptome analyses revealed similarities with native foetal bone marrow-derived MSCs. Significant KEGG pathways as well as GO terms are mostly related to immune function, embryonic skeletal system, and TGFβ-signalling. An AF-MSC-enriched gene set included putative AF-MSC markers PSG5, EMX-2, and EVR-3. In essence, C-section-derived AF-MSCs can be routinely obtained and are amenable for personalized cell therapies and disease modelling

Transplanted human pluripotent stem cell-derived mesenchymal stem cells support liver regeneration in Gunn rats

Gunn rats bear a mutation within the uridine diphosphate glucuronosyltransferase-1a1 (Ugt1a1) gene resulting in high serum bilirubin levels as seen in Crigler-Najjar syndrome. In this study, the Gunn rat was used as an animal model for heritable liver dysfunction. Induced mesenchymal stem cells (iMSCs) derived from embryonic stem cells (H1) and induced pluripotent stem cells were transplanted into Gunn rats after partial hepatectomy. The iMSCs engrafted and survived in the liver for up to 2 months. The transplanted iMSCs differentiated into functional hepatocytes as evidenced by partially suppressed hyperbilirubinemia and expression of multiple human-specific hepatocyte markers such as albumin, hepatocyte nuclear factor 4α, UGT1A1, cytokeratin 18, bile salt export pump, multidrug resistance protein 2, Na/taurocholate-cotransporting polypeptide, and α-fetoprotein. These findings imply that transplanted human iMSCs can contribute to liver regeneration in vivo and thus represent a promising tool for the treatment of inherited liver diseases