Stina Simonsson

Cartilage Tissue Engineering by the 3D Bioprinting of iPS Cells in a Nanocellulose/Alginate Bioink

Cartilage lesions can progress into secondary osteoarthritis and cause severe clinical problems in numerous patients. As a prospective treatment of such lesions, human-derived induced pluripotent stem cells (iPSCs) were shown to be 3D bioprinted into cartilage mimics using a nanofibrillated cellulose (NFC) composite bioink when co-printed with irradiated human chondrocytes. Two bioinks were investigated: NFC with alginate (NFC/A) or hyaluronic acid (NFC/HA). Low proliferation and phenotypic changes away from pluripotency were seen in the case of NFC/HA. However, in the case of the 3D-bioprinted NFC/A (60/40, dry weight % ratio) constructs, pluripotency was initially maintained, and after five weeks, hyaline-like cartilaginous tissue with collagen type II expression and lacking tumorigenic Oct4 expression was observed in 3D -bioprinted NFC/A (60/40, dry weight % relation) constructs. Moreover, a marked increase in cell number within the cartilaginous tissue was detected by 2-photon fluorescence microscopy, indicating the importance of high cell densities in the pursuit of achieving good survival after printing. We conclude that NFC/A bioink is suitable for bioprinting iPSCs to support cartilage production in co-cultures with irradiated chondrocytes.

Footprint-Free Human Induced Pluripotent Stem Cells From Articular Cartilage With Redifferentiation Capacity: A First Step Toward a Clinical-Grade Cell Source

Human induced pluripotent stem cells (iPSCs) are potential cell sources for regenerative medicine; however, clinical applications of iPSCs are restricted because of undesired genomic modifications associated with most reprogramming protocols. We show, for the first time, that chondrocytes from autologous chondrocyte implantation (ACI) donors can be efficiently reprogrammed into iPSCs using a nonintegrating method based on mRNA delivery, resulting in footprint‐free iPSCs (no genome‐sequence modifications), devoid of viral factors or remaining reprogramming molecules. The search for universal allogeneic cell sources for the ACI regenerative treatment has been difficult because making chondrocytes with high matrix‐forming capacity from pluripotent human embryonic stem cells has proven challenging and human mesenchymal stem cells have a predisposition to form hypertrophic cartilage and bone. We show that chondrocyte‐derived iPSCs can be redifferentiated in vitro into cartilage matrix‐producing cells better than fibroblast‐derived iPSCs and on par with the donor chondrocytes, suggesting the existence of a differentiation bias toward the somatic cell origin and making chondrocyte‐derived iPSCs a promising candidate universal cell source for ACI. Whole‐genome single nucleotide polymorphism array and karyotyping were used to verify the genomic integrity and stability of the established iPSC lines. Our results suggest that RNA‐based technology eliminates the risk of genomic integrations or aberrations, an important step toward a clinical‐grade cell source for regenerative medicine such as treatment of cartilage defects and osteoarthritis.

Phosphorylated Nucleolin Interacts with Translationally Controlled Tumor Protein during Mitosis and with Oct4 during Interphase in ES Cells

Background Reprogramming of somatic cells for derivation of either embryonic stem (ES) cells, by somatic cell nuclear transfer (SCNT), or ES-like cells, by induced pluripotent stem (iPS) cell procedure, provides potential routes toward non-immunogenic cell replacement therapies. Nucleolar proteins serve as markers for activation of embryonic genes, whose expression is crucial for successful reprogramming. Although Nucleolin (Ncl) is one of the most abundant nucleolar proteins, its interaction partners in ES cells have remained unidentified. Methodology Here we explored novel Ncl-interacting proteins using in situ proximity ligation assay (PLA), colocalization and immunoprecipitation (IP) in ES cells. Principal Findings We found that phosphorylated Ncl (Ncl-P) interacted with translationally controlled tumor protein (Tpt1) in murine ES cells. The Ncl-P/Tpt1 complex peaked during mitosis and was reduced upon retinoic acid induced differentiation, signifying a role in cell proliferation. In addition, we showed that Ncl-P interacted with the transcription factor Oct4 during interphase in human as well as murine ES cells, indicating of a role in transcription. The Ncl-P/Oct4 complex peaked during early stages of spontaneous human ES cell differentiation and may thus be involved in the initial differentiation event(s) of mammalian development. Conclusions Here we described two novel protein-protein interactions in ES cells, which give us further insight into the complex network of interacting proteins in pluripotent cells.

Translationally controlled tumor protein interacts with nucleophosmin during mitosis in ES cells

Somatic cell nuclear transfers and the generation of induced pluripotent stem cells provide potential routes towards non-immunogenic cell replacement therapies. Translationally controlled tumor protein (Tpt1) was recently suggested to regulate cellular pluripotency. Here we explore functions of Tpt1 in mouse embryonic stem (ES) cells. We find that Tpt1 is present in the nucleus and cytoplasm of ES cells, and that specifically nuclear Tpt1 decreases upon cell differentiation. We also find that endogenous Tpt1 forms a complex with endogenous nucleophosmin/nucleoplasmin family member 1 (Npm1) in a cell cycle dependent manner. The Tpt1-Npm1 complex peaks sharply during mitosis and is independent of phosphorylation by Polo-like kinase. Differentiation by retinoic acid decreases Tpt1-Npm1 complex levels. Moreover, Tpt1 knock-down or over-expression reduces proliferation whereas Npm1 over-expression increases proliferation in ES cells. Cells depleted for both Tpt1 and Npm1 exhibit significantly reduced proliferation compared to cells depleted for Tpt1 alone, whereas cells over-expressing both Tpt1 and Npm1 show normal proliferation. Our findings reveal a role for the Tpt1-Npm1 complex in cell proliferation and identify the Tpt1-Npm1 complex as a potential biomarker for mitotic ES cells.

Complementary intrastrand base pairing during initiation of Herpes simplex virus type 1 DNA replication

The herpes simplex virus type 1 origin of DNA replication, oriS, contains three copies of the recognition sequence for the viral initiator protein, origin binding protein (OBP), arranged in two palindromes. The central box I forms a short palindrome with box III and a long palindrome with box II. Single-stranded oriS adopts a conformation, oriS*, that is tightly bound by OBP. Here we demonstrate that OBP binds to a box III–box I hairpin with a 3′ single-stranded tail in oriS*. Mutations designed to destabilize the hairpin abolish the binding of OBP to oriS*. The same mutations also inhibit DNA replication. Second site complementary mutations restore binding of OBP to oriS* as well as the ability of mutated oriS to support DNA replication. OriS* is also an efficient activator of the hydrolysis of ATP by OBP. Sequence analyses show that a box III–box I palindrome is an evolutionarily conserved feature of origins of DNA replication from human, equine, bovine, and gallid alpha herpes viruses. We propose that oriS facilitates initiation of DNA synthesis in two steps and that OBP exhibits exquisite specificity for the different conformations oriS adopts at these stages. Our model suggests that distance-dependent cooperative binding of OBP to boxes I and II in duplex DNA is succeeded by specific recognition of a box III–box I hairpin in partially unwound DNA.

The Herpes Simplex Virus Type 1 Origin Binding Protein SPECIFIC RECOGNITION OF PHOSPHATES AND METHYL GROUPS DEFINES THE INTERACTING SURFACE FOR A MONOMERIC DNA BINDING DOMAIN IN THE MAJOR GROOVE OF DNA

The UL9 gene of herpes simplex virus type 1 (HSV-1) encodes an origin binding protein (OBP). It is an ATP-dependent DNA helicase and a sequence-specific DNA-binding protein. The latter function is carried out by the C-terminal domain of OBP (ΔOBP). We have now performed a quantitative analysis of the interaction between ΔOBP and its recognition sequence, GTTCGCAC, in oriS. Initially optimal conditions for binding were carefully determined. We observed that complexes with different electrophoretic mobilities were formed. A cross-linking experiment demonstrated that nonspecific complexes containing 2 or more protein monomers per DNA molecule were formed at high protein concentrations. The specific complex formed at low concentrations of ΔOBP had an electrophoretic mobility corresponding to a 1:1 complex. We then demonstrated that the methyl groups of thymine in the major groove were essential for high affinity binding. Changes in the minor groove had considerably smaller effects. Ethylation interference experiments indicated that specific contacts were made between OBP and three phosphates in the recognition sequence. Finally, these observations were used to present a model of the surface of DNA that interacts with ΔOBP in a sequence-specific manner.

SAF-A Has a Role in Transcriptional Regulation of Oct4 in ES Cells Through Promoter Binding

Methodologies to reprogram somatic cells into patient-specific pluripotent cells, which could potentially be used in personalized drug discovery and cell replacement therapies, are currently under development. Oct4 activation is essential for successful reprogramming and pluripotency of embryonic stem (ES) cells, albeit molecular details of Oct4 activation are not completely understood. Here we report that endogenous SAF-A is involved in regulation of Oct4 expression, binds the Oct4 proximal promoter in ES cells, and dissociates from the promoter upon early differentiation induced by LIF withdrawal. Depletion of SAF-A decreases Oct4 expression even in the presence of LIF, and results in an increase of the mesodermal marker Brachyury. The overexpression of wild-type human SAF-A rescues the mouse knock-down phenotype and results in increased Oct4 level. We also demonstrate that endogenous SAF-A interacts with the C-terminal domain (CTD) of endogenous RNA polymerase II and that the interaction is independent of CTD phosphorylation and mRNA. Moreover, we show that SAF-A exist in complexes with transcription factors Sox2 and Oct4 as well as STAT3 in ES cells. The number of endogenous SAF-A:Oct4 and SAF-A:Sox2 complexes decreases upon LIF depletion. These discoveries allow us to propose a model for activation of Oct4 transcription.

Amyloid precursor protein expression and processing are differentially regulated during cortical neuron differentiation

Amyloid precursor protein (APP) and its cleavage product amyloid β (Aβ) have been thoroughly studied in Alzheimer’s disease. However, APP also appears to be important for neuronal development. Differentiation of induced pluripotent stem cells (iPSCs) towards cortical neurons enables in vitro mechanistic studies on human neuronal development. Here, we investigated expression and proteolytic processing of APP during differentiation of human iPSCs towards cortical neurons over a 100-day period. APP expression remained stable during neuronal differentiation, whereas APP processing changed. α-Cleaved soluble APP (sAPPα) was secreted early during differentiation, from neuronal progenitors, while β-cleaved soluble APP (sAPPβ) was first secreted after deep-layer neurons had formed. Short Aβ peptides, including Aβ1-15/16, peaked during the progenitor stage, while processing shifted towards longer peptides, such as Aβ1-40/42, when post-mitotic neurons appeared. This indicates that APP processing is regulated throughout differentiation of cortical neurons and that amyloidogenic APP processing, as reflected by Aβ1-40/42, is associated with mature neuronal phenotypes.

Developmental studies of Xenopus shelterin complexes: the message to reset telomere length is already present in the egg

The 6‐protein complex shelterin protects the telomeres of human chromosomes. The recent discovery that telomeres are important for epigenetic gene regulation and vertebrate embryonic development calls for the establishment of model organisms to study shelterin and telomere function under normal developmental conditions. Here, we report the sequences of the shelterin‐encoding genes in Xenopus laevis and its close relation Xenopus tropicalis. In vitro expression and biochemical characterization of the Xenopus shelterin proteins TRF1, TRF2, POT1, TIN2, RAP1, TPP1, and the shelterin accessory factor PINX1 indicate that all main functions of their human orthologs are conserved in Xenopus. The XlTRF1 and XtTRF1 proteins bind double‐stranded telomeric DNA sequence specifically and interact with XlTIN2 and XtTIN2, respectively. Similarly, the XlTRF2 and XtTRF2 proteins bind double‐stranded telomeric DNA and interact with XlRAP1 and XtRAP1, respectively, whereas the XlPOT1 and XtPOT1 proteins bind singlestranded telomeric DNA. Real‐time PCR further reveals the gene expression profiles for telomerase and the shelterin genes during embryogenesis. Notably, the composition of shelterin and the formation of its subcomplexes appear to be temporally regulated during embryonic development. Moreover, unexpectedly high telomerase and shelterin gene expression during early embryogenesis may reflect a telomere lengthresetting mechanism, similar to that reported for induced pluripotent stem cells and for animals cloned through somatic nuclear transfer.— Vizlin‐Hodzic, D.,Ryme, J., Simonsson, S., Simonsson, T. Developmental studies of Xenopus shelterin complexes: the message to reset telomere length is already present in the egg. FASEBJ. 23, 2587–2594 (2009)

Highly Synchronized Expression of Lineage-Specific Genes during In Vitro Hepatic Differentiation of Human Pluripotent Stem Cell Lines

Human pluripotent stem cells- (hPSCs-) derived hepatocytes have the potential to replace many hepatic models in drug discovery and provide a cell source for regenerative medicine applications. However, the generation of fully functional hPSC-derived hepatocytes is still a challenge. Towards gaining better understanding of the differentiation and maturation process, we employed a standardized protocol to differentiate six hPSC lines into hepatocytes and investigated the synchronicity of the hPSC lines by applying RT-qPCR to assess the expression of lineage-specific genes (OCT4, NANOG, T, SOX17, CXCR4, CER1, HHEX, TBX3, PROX1, HNF6, AFP, HNF4a, KRT18, ALB, AAT, and CYP3A4) which serve as markers for different stages during liver development. The data was evaluated using correlation and clustering analysis, demonstrating that the expression of these markers is highly synchronized and correlated well across all cell lines. The analysis also revealed a distribution of the markers in groups reflecting the developmental stages of hepatocytes. Functional analysis of the differentiated cells further confirmed their hepatic phenotype. Taken together, these results demonstrate, on the molecular level, the highly synchronized differentiation pattern across multiple hPSC lines. Moreover, this study provides additional understanding for future efforts to improve the functionality of hPSC-derived hepatocytes and thereby increase the value of related models.