Tanja Ilmarinen

Comparative Analysis of Targeted Differentiation of Human Induced Pluripotent Stem Cells (hiPSCs) and Human Embryonic Stem Cells Reveals Variability Associated With Incomplete Transgene Silencing in Retrovirally Derived hiPSC Lines

Functional hepatocytes, cardiomyocytes, neurons, and retinal pigment epithelial (RPE) cells derived from human embryonic stem cells (hESCs) or human induced pluripotent stem cells (hiPSCs) could provide a defined and renewable source of human cells relevant for cell replacement therapies, drug discovery, toxicology testing, and disease modeling. In this study, we investigated the differences between the differentiation potentials of three hESC lines, four retrovirally derived hiPSC lines, and one hiPSC line derived with the nonintegrating Sendai virus technology. Four independent protocols were used for hepatocyte, cardiomyocyte, neuronal, and RPE cell differentiation. Overall, cells differentiated from hESCs and hiPSCs showed functional similarities and similar expression of genes characteristic of specific cell types, and differences between individual cell lines were also detected. Reactivation of transgenic OCT4 was detected specifically during RPE differentiation in the retrovirally derived lines, which may have affected the outcome of differentiation with these hiPSCs. One of the hiPSC lines was inferior in all directions, and it failed to produce hepatocytes. Exogenous KLF4 was incompletely silenced in this cell line. No transgene expression was detected in the Sendai virus‐derived hiPSC line. These findings highlight the problems related to transgene expression in retrovirally derived hiPSC lines.

Small-Molecule Induction Promotes Corneal Epithelial Cell Differentiation from Human Induced Pluripotent Stem Cells

Summary Human induced pluripotent stem cells (hiPSCs) offer unique opportunities for developing novel cell-based therapies and disease modeling. In this study, we developed a directed differentiation method for hiPSCs toward corneal epithelial progenitor cells capable of terminal differentiation toward mature corneal epithelial-like cells. In order to improve the efficiency and reproducibility of our method, we replicated signaling cues active during ocular surface ectoderm development with the help of two small-molecule inhibitors in combination with basic fibroblast growth factor (bFGF) in serum-free and feeder-free conditions. First, small-molecule induction downregulated the expression of pluripotency markers while upregulating several transcription factors essential for normal eye development. Second, protein expression of the corneal epithelial progenitor marker p63 was greatly enhanced, with up to 95% of cells being p63 positive after 5 weeks of differentiation. Third, corneal epithelial-like cells were obtained upon further maturation.

Ultrathin Polyimide Membrane as Cell Carrier for Subretinal Transplantation of Human Embryonic Stem Cell Derived Retinal Pigment Epithelium.

In this study, we investigated the suitability of ultrathin and porous polyimide (PI) membrane as a carrier for subretinal transplantation of human embryonic stem cell (hESC) -derived retinal pigment epithelial (RPE) cells in rabbits. The in vivo effects of hESC-RPE cells were analyzed by subretinal suspension injection into Royal College of Surgeons (RCS) rats. Rat eyes were analyzed with electroretinography (ERG) and histology. After analyzing the surface and permeability properties of PI, subretinal PI membrane transplantations with and without hESC-RPE were performed in rabbits. The rabbits were followed for three months and eyes analyzed with fundus photography, ERG, optical coherence tomography (OCT), and histology. Animals were immunosuppressed with cyclosporine the entire follow-up time. In dystrophic RCS rats, ERG and outer nuclear layer (ONL) thickness showed some rescue after hESC-RPE injection. Cells positive for human antigen were found in clusters under the retina 41 days post-injection but not anymore after 105 days. In rabbits, OCT showed good placement of the PI. However, there was loss of pigmentation on the hESC-RPE-PI over time. In the eyes with PI alone, no obvious signs of inflammation or retinal atrophy were observed. In the presence of hESC-RPE, mononuclear cell infiltration and retinal atrophy were observed around the membranes. The porous ultrathin PI membrane was well-tolerated in the subretinal space and is a promising scaffold for RPE transplantation. However, the rejection of the transplanted cells seems to be a major problem and the given immunosuppression was insufficient for reduction of xenograft induced inflammation.

Towards a defined, serum‐ and feeder‐free culture of stratified human oral mucosal epithelium for ocular surface reconstruction

Purpose:  Ocular surface reconstruction with cultivated oral mucosal epithelial transplantation technique is a viable treatment option for severe ocular surface injuries and diseases with limbal stem cell deficiency. Currently, this technique is based on utilization of xenogenic, allogenic or undefined components such as murine 3T3 feeders, serum and amniotic membrane. In this study, we aimed to find a more defined culture method to generate stratified human oral mucosal epithelium.

Human pluripotent stem cell-derived limbal epithelial stem cells on bioengineered matrices for corneal reconstruction

Corneal epithelium is renewed by limbal epithelial stem cells (LESCs), a type of tissue-specific stem cells located in the limbal palisades of Vogt at the corneo-scleral junction. Acute trauma or inflammatory disorders of the ocular surface can destroy these stem cells, leading to limbal stem cell deficiency (LSCD) - a painful and vision-threatening condition. Treating these disorders is often challenging and complex, especially in bilateral cases with extensive damage. Human pluripotent stem cells (hPSCs) provide new opportunities for corneal reconstruction using cell-based therapy. Here, we investigated the use of hPSC-derived LESC-like cells on bioengineered collagen matrices in serum-free conditions, aiming for clinical applications to reconstruct the corneal epithelium and partially replace the damaged stroma. Differentiation of hPSCs towards LESC-like cells was directed using small-molecule induction followed by maturation in corneal epithelium culture medium. After four to five weeks of culture, differentiated cells were seeded onto bioengineered matrices fabricated as transparent membranes of uniform thickness, using medical-grade porcine collagen type I and a hybrid cross-linking technology. The bioengineered matrices were fully transparent, with high water content and swelling capacity, and parallel lamellar microstructure. Cell proliferation of hPSC-LESCs was significantly higher on bioengineered matrices than on collagen-coated control wells after two weeks of culture, and LESC markers p63 and cytokeratin 15, along with proliferation marker Ki67 were expressed even after 30 days in culture. Overall, hPSC-LESCs retained their capacity to self-renew and proliferate, but were also able to terminally differentiate upon stimulation, as suggested by protein expression of cytokeratins 3 and 12. We propose the use of bioengineered collagen matrices as carriers for the clinically-relevant hPSC-derived LESC-like cells, as a novel tissue engineering approach for corneal reconstruction.

Patient-Specific Induced Pluripotent Stem Cell-Derived RPE Cells: Understanding the Pathogenesis of Retinopathy in Long-Chain 3-Hydroxyacyl-CoA Dehydrogenase Deficiency.

Purpose: Retinopathy is an important manifestation of trifunctional protein (TFP) deficiencies but not of other defects of fatty acid oxidation. The common homozygous mutation in the TFP alpha subunit gene HADHA (hydroxyacyl-CoA dehydrogenase), c.1528G>C, affects the long-chain 3-hydroxyacyl-CoA dehydrogenase (LCHAD) activity of TFP, and blindness in infancy. The pathogenesis of the retinopathy is unknown. This study aims to utilize human induced pluripotent stem cell (hiPSC) technology to create a disease model for the disorder, and to derive clues for retinopathy pathogenesis. Methods: We implemented hiPSC technology to generate LCHAD deficiency (LCHADD) patient specific retinal pigment epithelial (RPE) monolayers. These patient and control RPEs were extensively characterised for function and structure, as well as for lipid composition by mass spectrometry. Results: The hiPSC derived RPE monolayers of patients and controls were functional, as they both were able to phagocytose the photoreceptor outer segments in vitro. Interestingly, the patient RPEs had intense cytoplasmic neutral lipid accumulation and lipidomic analysis revealed an increased triglyceride accumulation. Further, patient RPEs were small and irregular in shape, and their tight junctions were disorganized. Their ultrastructure showed decreased pigmentation, few melanosomes, and more melanolysosomes. Conclusion: We demonstrate that RPE cell model reveals novel early pathogenic changes in LCHADD retinopathy, with robust lipid accumulation, inefficient pigmentation that is evident soon after differentiation, and a defect in forming tight junctions inducing apoptosis. We propose that LCHADD-RPEs are an important model for mitochondrial TFP retinopathy, and that their early pathogenic changes contribute to infantile blindness of LCHADD.PURPOSE Retinopathy is an important manifestation of trifunctional protein (TFP) deficiencies but not of other defects of fatty acid oxidation. The common homozygous mutation in the TFP α-subunit gene HADHA (hydroxyacyl-CoA dehydrogenase), c.1528G>C, affects the long-chain 3-hydroxyacyl-CoA dehydrogenase (LCHAD) activity of TFP and blindness in infancy. The pathogenesis of the retinopathy is unknown. This study aimed to utilize human induced pluripotent stem cell (hiPSC) technology to create a disease model for the disorder, and to derive clues for retinopathy pathogenesis. METHODS We implemented hiPSC technology to generate LCHAD deficiency (LCHADD) patient-specific retinal pigment epithelial (RPE) monolayers. These patient and control RPEs were extensively characterized for function and structure, as well as for lipid composition by mass spectrometry. RESULTS The hiPSC-derived RPE monolayers of patients and controls were functional, as they both were able to phagocytose the photoreceptor outer segments in vitro. Interestingly, the patient RPEs had intense cytoplasmic neutral lipid accumulation, and lipidomic analysis revealed an increased triglyceride accumulation. Further, patient RPEs were small and irregular in shape, and their tight junctions were disorganized. Their ultrastructure showed decreased pigmentation, few melanosomes, and more melanolysosomes. CONCLUSIONS We demonstrate that the RPE cell model reveals novel early pathogenic changes in LCHADD retinopathy, with robust lipid accumulation, inefficient pigmentation that is evident soon after differentiation, and a defect in forming tight junctions inducing apoptosis. We propose that LCHADD-RPEs are an important model for mitochondrial TFP retinopathy, and that their early pathogenic changes contribute to infantile blindness of LCHADD.

Comparative proteomics reveals human pluripotent stem cell-derived limbal epithelial stem cells are similar to native ocular surface epithelial cells

Limbal epithelial stem cells (LESCs) are tissue-specific stem cells responsible for renewing the corneal epithelium. Acute trauma or chronic disease affecting LESCs may disrupt corneal epithelial renewal, causing vision threatening and painful ocular surface disorders, collectively referred to as LESC deficiency (LESCD). These disorders cannot be treated with traditional corneal transplantation and therefore alternative cell sources for successful cell-based therapy are needed. LESCs derived from human pluripotent stem cells (hPSCs) are a prospective source for ocular surface reconstruction, yet critical evaluation of these cells is crucial before considering clinical applications. In order to quantitatively evaluate hPSC-derived LESCs, we compared protein expression in native human corneal cells to that in hPSC-derived LESCs using isobaric tag for relative and absolute quantitation (iTRAQ) technology. We identified 860 unique proteins present in all samples, including proteins involved in cell cycling, proliferation, differentiation and apoptosis, various LESC niche components, and limbal and corneal epithelial markers. Protein expression profiles were nearly identical in LESCs derived from two different hPSC lines, indicating that the differentiation protocol is reproducible, yielding homogeneous cell populations. Their protein expression profile suggests that hPSC-derived LESCs are similar to the human ocular surface epithelial cells, and possess LESC-like characteristics.

Langmuir-Schaefer film deposition onto honeycomb porous films for retinal tissue engineering

Age-related macular degeneration (AMD) is the leading cause of vision loss in senior citizens in the developed world. The disease is characterised by the degeneration of a specific cell layer at the back of the eye - the retinal pigment epithelium (RPE), which is essential in retinal function. The most promising therapeutic option to restore the lost vision is considered to be RPE cell transplantation. This work focuses on the development of biodegradable biomaterials with similar properties to the native Bruchs membrane as carriers for RPE cells. In particular, the breath figure (BF) method was used to create semi-permeable microporous films, which were thereafter used as the substrate for the consecutive Langmuir-Schaefer (LS) deposition of highly organised layers of collagen type I and collagen type IV. The newly developed biomaterials were further characterised in terms of surface porosity, roughness, hydrophilicity, collagen distribution, diffusion properties and hydrolytic stability. Human embryonic stem cell-derived RPE cells (hESC-RPE) cultured on the biomaterials showed good adhesion, spreading and morphology, as well as the expression of specific protein markers. Cell function was additionally confirmed by the assessment of the phagocytic capacity of hESC-RPE. Throughout the study, microporous films consistently showed better results as cell culture materials for hESC-RPE than dip-coated controls. This work demonstrates the potential of the BF-LS combined technologies to create biomimetic prosthetic Bruchs membranes for hESC-RPE transplantation. STATEMENT OF SIGNIFICANCE Age-related macular degeneration (AMD) is a leading cause of central blindness in developed countries, associated with the degeneration of the retinal pigment epithelium (RPE), a specific cell layer at the back of the eye. Transplantation of RPE cells derived from stem cells is considered the best option to treat these patients. In this work, we developed a cell carrier for human embryonic stem cell-derived RPE that resembled the upper layers of the membrane that naturally supports the RPE cells in the retina. The new combination of technologies employed in this study resulted in very promising materials as confirmed by our studies on cell proliferation, morphology and function.

Honeycomb porous films as permeable scaffold materials for human embryonic stem cell‐derived retinal pigment epithelium

Age-related macular degeneration (AMD) is a leading cause of blindness in developed countries, characterised by the degeneration of the retinal pigment epithelium (RPE), a pigmented cell monolayer that closely interacts with the photoreceptors. RPE transplantation is thus considered a very promising therapeutic option to treat this disease. In this work, porous honeycomb-like films are for the first time investigated as scaffold materials for human embryonic stem cell-derived retinal pigment epithelium (hESC-RPE). By changing the conditions during film preparation, it was possible to produce films with homogeneous pore distribution and adequate pore size (∼3-5 µm), that is large enough to ensure high permeability but small enough to enable cell adherence and spreading. A brief dip-coating procedure with collagen type IV enabled the homogeneous adsorption of the protein to the walls and bottom of pores, increasing the hydrophilicity of the surface. hESC-RPE adhered and proliferated on all the collagen-coated materials, regardless of small differences in pore size. The differentiation of hESC-RPE was confirmed by the detection of specific RPE protein markers. These results suggest that the porous honeycomb films can be promising candidates for hESC-RPE tissue engineering, importantly enabling the free flow of ions and molecules across the material.

Hydrazone crosslinked hyaluronan-based hydrogels for therapeutic delivery of adipose stem cells to treat corneal defects

Corneal blindness is a worldwide problem, plagued by insufficient amount of high-quality donor tissue. Cell therapy using human adipose stem cells (hASCs) has risen as an alternative to regenerate damaged corneal stromal tissue, the main structural and refractive layer of the cornea. Herein we propose a method to deliver hASCs into corneal defects in hyaluronan (HA)-based hydrogels, which form rapidly in situ by hydrazone crosslinking. We fabricated two different HA-based hydrazone-crosslinked hydrogels (HALD1-HACDH and HALD2-HAADH), and characterized their swelling, degradation, mechanical, rheological and optical properties and their ability to support hASC survival. To promote hASC attachment and survival, we incorporated collagen I (col I) to the more stable HALD1-HACDH hydrogel, since the HALD2-HAADH hydrogel suffered swift degradation in culture conditions. We then used an organ culture model with excised porcine corneas to study the delivery of hASCs in these three hydrogels for stromal defect repair. Although all hydrogels showed good hASC survival directly after encapsulation, only the collagen-containing HALD1-HACDH-col I hydrogel showed cells with elongated morphology, and significantly higher cell metabolic activity than the HALD1-HACDH gel. The addition of col I also increased the stiffness and reduced the swelling ratio of the resulting hydrogel. Most importantly, the corneal organ culture model demonstrated these hydrogels as clinically feasible cell delivery vehicles to corneal defects, allowing efficient hASC integration to the corneal stroma and overgrowth of corneal epithelial cells.