Questionnaire Study to Investigate the Preferences of Children, Parents, and Healthcare Professionals for Different Formulations of Oral Medicinal Products.

Since the acceptability of a medicine can significantly impact therapeutic outcomes, this study aimed to determine and compare the preferences of children, parents, and healthcare professionals for the most commonly used pediatric oral medicine formulations (syrup, mini-tablets, oblong tablets, round tablets) addressing all pediatric age groups, 0-<18 years (y). This survey study employed sex-, age-, and participant group-adapted questionnaires for eight cohorts of participants, i.e., children 6-<12 y, adolescents 12-<18 y, parents of children in four age groups (0-<2 y, 2-<6 y, 6-<12 y, and 12-<18 y), nurses, and pediatricians. Descriptive statistics were used for data analysis. In the age groups 0-<2 y and 2-<6 y, mini-tablets were preferred over syrup by all participants. In the age group 6-12 y, solid dosage forms were also preferred over syrup by all participants. In the age group 12-<18 y, healthcare professionals preferred solid dosage forms over syrup. Parents preferred higher amounts of mini-tablets and syrup compared to round and oblong tablets, while adolescents' preferences did not differentiate between these formulations. Based on the study results and in contrast to current practice, it is suggested to consider solid dosage forms for future age-appropriate medicinal products already for younger age groups.

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.

Human Induced Pluripotent Stem Cell-Derived Mesenchymal Stem Cells Acquire Rejuvenation and Reduced Heterogeneity.

Despite the uniform selection criteria for the isolation of human mesenchymal stem cells (MSCs), considerable heterogeneity exists which reflects the distinct tissue origins and differences between individuals with respect to their genetic background and age. This heterogeneity is manifested by the variabilities seen in the transcriptomes, proteomes, secretomes, and epigenomes of tissue-specific MSCs. Here, we review literature on different aspects of MSC heterogeneity including the role of epigenetics and the impact of MSC heterogeneity on therapies. We then combine this with a meta-analysis of transcriptome data from distinct MSC subpopulations derived from bone marrow, adipose tissue, cruciate, tonsil, kidney, umbilical cord, fetus, and induced pluripotent stem cells derived MSCs (iMSCs). Beyond that, we investigate transcriptome differences between tissue-specific MSCs and pluripotent stem cells. Our meta-analysis of numerous MSC-related data sets revealed markers and associated biological processes characterizing the heterogeneity and the common features of MSCs from various tissues. We found that this heterogeneity is mainly related to the origin of the MSCs and infer that microenvironment and epigenetics are key drivers. The epigenomes of MSCs alter with age and this has a profound impact on their differentiation capabilities. Epigenetic modifications of MSCs are propagated during cell divisions and manifest in differentiated cells, thus contributing to diseased or healthy phenotypes of the respective tissue. An approach used to reduce heterogeneity caused by age- and tissue-related epigenetic and microenvironmental patterns is the iMSC concept: iMSCs are MSCs generated from induced pluripotent stem cells (iPSCs). During iMSC generation epigenetic and chromatin remodeling result in a gene expression pattern associated with rejuvenation thus allowing to overcome age-related shortcomings (e.g., limited differentiation and proliferation capacity). The importance of the iMSC concept is underlined by multiple clinical trials. In conclusion, we propose the use of rejuvenated iMSCs to bypass tissue- and age-related heterogeneity which are associated with native MSCs.

The FGF, TGFß and WNT axis Modulate Self-renewal of Human SIX2+ Urine Derived Renal Progenitor Cells.

Human urine is a non-invasive source of renal stem cells with regeneration potential. Urine-derived renal progenitor cells were isolated from 10 individuals of both genders and distinct ages. These renal progenitors express pluripotency-associated proteins- TRA-1-60, TRA-1-81, SSEA4, C-KIT and CD133, as well as the renal stem cell markers -SIX2, CITED1, WT1, CD24 and CD106. The transcriptomes of all SIX2+ renal progenitors clustered together, and distinct from the human kidney biopsy-derived epithelial proximal cells (hREPCs). Stimulation of the urine-derived renal progenitor cells (UdRPCs) with the GSK3ß-inhibitor (CHIR99021) induced differentiation. Transcriptome and KEGG pathway analysis revealed upregulation of WNT-associated genes- AXIN2, JUN and NKD1. Protein interaction network identified JUN- a downstream target of the WNT pathway in association with STAT3, ATF2 and MAPK1 as a putative negative regulator of self-renewal. Furthermore, like pluripotent stem cells, self-renewal is maintained by FGF2-driven TGFß-SMAD2/3 pathway. The urine-derived renal progenitor cells and the data presented should lay the foundation for studying nephrogenesis in human.

Human iPSC-derived iMSCs improve bone regeneration in mini-pigs.

Autologous bone marrow concentrate (BMC) and mesenchymal stem cells (MSCs) have beneficial effects on the healing of bone defects. To address the shortcomings associated with the use of primary MSCs, induced pluripotent stem cell (iPSC)-derived MSCs (iMSCs) have been proposed as an alternative. The aim of this study was to investigate the bone regeneration potential of human iMSCs combined with calcium phosphate granules (CPG) in critical-size defects in the proximal tibias of mini-pigs in the early phase of bone healing compared to that of a previously reported autograft treatment and treatment with a composite made of either a combination of autologous BMC and CPG or CPG alone. iMSCs were derived from iPSCs originating from human fetal foreskin fibroblasts (HFFs). They were able to differentiate into osteoblasts in vitro, express a plethora of bone morphogenic proteins (BMPs) and secrete paracrine signaling-associated cytokines such as PDGF-AA and osteopontin. Radiologically and histomorphometrically, HFF-iMSC + CPG transplantation resulted in significantly better osseous consolidation than the transplantation of CPG alone and produced no significantly different outcomes compared to the transplantation of autologous BMC + CPG after 6 weeks. The results of this translational study imply that iMSCs represent a valuable future treatment option for load-bearing bone defects in humans.

Characterization of burn wound healing gel prepared from human amniotic membrane and Aloe vera extract.

BACKGROUND: Skin burn wound is a notable medical burden worldwide. Rapid and effective treatment of burnt skin is vital to fasten wound closure and healing properly. Amniotic graft and Aloe vera are widely used as wound managing biomaterials. Sophisticated processing, high cost, availability, and the requirement of medics for transplantation limit the application of amnion grafts. We aim to prepare a novel gel from amnion combined with the Aloe vera extract for burn wound healing which overcome the limitations of graft. METHODS: Two percent human amniotic membrane (AM), Aloe vera (AV) and AM+AV gels were prepared. In vitro cytotoxicity, biocompatibility, cell attachment, proliferation, wound healing scratch assays were performed in presence of the distinct gels. After skin irritation study, second-degree burns were induced on dorsal region of Wistar rats; and gels were applied to observe the healing potential in vivo. Besides, macroscopical measurement of wound contraction and re-epithelialization; gel treated skin was histologically investigated by Hematoxylin and eosin (H&E) staining. Finally, quantitative assessment of angiogenesis, inflammation, and epithelialization was done. RESULTS: The gels were tested to be non-cytotoxic to nauplii and compatible with human blood and skin cells. Media containing 500 µg/mL AM+AV gel were observed to promote HaCaT and HFF1 cells attachment and proliferation. In vitro scratch assay demonstrated that AM+AV significantly accelerated wound closure through migration of HaCaT cells. No erythema and edema were observed in skin irritation experiments confirming the applicability of the gels. AV and AM+AV groups showed significantly accelerated wound closure through re-epithelialization and wound contraction with P < 0.01. Macroscopically, AM and AM+AV treated wound recovery rates were 87 and 90% respectively with P < 0.05. Histology analysis revealed significant epitheliazation and angiogenesis in AM+AV treated rats compared to control (P < 0.05). AM+AV treated wounds had thicker regenerated epidermis, increased number of blood vessels, and greater number of proliferating keratinocytes within the epidermis. CONCLUSION: We demonstrated that a gel consisting of a combination of amnion and Aloe vera extract has high efficacy as a burn wound healing product. Amniotic membrane combined with the carrier Aloe vera in gel format is easy to produce and to apply.

Constructing an Isogenic 3D Human Nephrogenic Progenitor Cell Model Composed of Endothelial, Mesenchymal, and SIX2-Positive Renal Progenitor Cells.

Urine has become the source of choice for noninvasive renal epithelial cells and renal stem cells which can be used for generating induced pluripotent stem cells. The aim of this study was to generate a 3D nephrogenic progenitor cell model composed of three distinct cell types-urine-derived SIX2-positive renal progenitor cells, iPSC-derived mesenchymal stem cells, and iPSC-derived endothelial cells originating from the same individual. Characterization of the generated mesenchymal stem cells revealed plastic adherent growth and a trilineage differentiation potential to adipocytes, chondrocytes, and osteoblasts. Furthermore, these cells express the typical MSC markers CD73, CD90, and CD105. The induced endothelial cells express the endothelial cell surface marker CD31. Upon combination of urine-derived renal progenitor cells, induced mesenchymal stem cells, and induced endothelial cells at a set ratio, the cells self-condensed into three-dimensional nephrogenic progenitor cells which we refer to as 3D-NPCs. Immunofluorescence-based stainings of sectioned 3D-NPCs revealed cells expressing the renal progenitor cell markers (SIX2 and PAX8), podocyte markers (Nephrin and Podocin), the endothelial marker (CD31), and mesenchymal markers (Vimentin and PDGFR-ß). These 3D-NPCs share kidney progenitor characteristics and thus the potential to differentiate into podocytes and proximal and distal tubules. As urine-derived renal progenitor cells can be easily obtained from cells shed into urine, the generation of 3D-NPCs directly from renal progenitor cells instead of pluripotent stem cells or kidney biopsies holds a great potential for the use in nephrotoxicity tests, drug screening, modelling nephrogenesis and diseases.

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.

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.

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.

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

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.

Human mesenchymal factors induce rat hippocampal- and human neural stem cell dependent oligodendrogenesis.

The generation of new oligodendrocytes is essential for adult brain repair in diseases such as multiple sclerosis. We previously identified the multifunctional p57kip2 protein as a negative regulator of myelinating glial cell differentiation and as an intrinsic switch of glial fate decision in adult neural stem cells (aNSCs). In oligodendroglial precursor cells (OPCs), p57kip2 protein nuclear exclusion was recently found to be rate limiting for differentiation to proceed. Furthermore, stimulation with mesenchymal stem cell (MSC)-derived factors enhanced oligodendrogenesis by yet unknown mechanisms. To elucidate this instructive interaction, we investigated to what degree MSC secreted factors are species dependent, whether hippocampal aNSCs respond equally well to such stimuli, whether apart from oligodendroglial differentiation also tissue integration and axonal wrapping can be promoted and whether the oligodendrogenic effect involved subcellular translocation of p57kip2. We found that CC1 positive oligodendrocytes within the hilus express nuclear p57kip2 protein and that MSC dependent stimulation of cultured hippocampal aNSCs was not accompanied by nuclear p57kip2 exclusion as observed for parenchymal OPCs after spontaneous differentiation. Stimulation with human MSC factors was observed to equally promote rat stem cell oligodendrogenesis, axonal wrapping and tissue integration. As forced nuclear shuttling of p57kip2 led to decreased CNPase- but elevated GFAP expression levels, this indicates heterogenic oligodendroglial mechanisms occurring between OPCs and aNSCs. We also show for the first time that dominant pro-oligodendroglial factors derived from human fetal MSCs can instruct human induced pluripotent stem cell-derived NSCs to differentiate into O4 positive oligodendrocytes.

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 TGFß-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.

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 51year 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 (TGFß, MEK and GSK3ß) 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.