Scientists do not fully understand whether these cells are actually stem cells or what types of cells they are capable of generating. They do agree that not all MSCs are the same, and that their characteristics depend on where in the body they come from and how they are isolated and grown. IPS cells are critical tools to help scientists learn more about normal development and disease onset and progression, and they are also useful for developing and testing new drugs and therapies.
Scientists are exploring what these differences are and what they mean. For one thing, the first iPS cells were produced by using viruses to insert extra copies of genes into tissue-specific cells.
Induced Pluripotent Stem Cells (iPSCs)
Researchers are experimenting with many alternative ways to create iPS cells so that they can ultimately be used as a source of cells or tissues for medical treatments. Stem cells Stem cells are the foundation for every organ and tissue in your body. In this section: Embryonic stem cells Tissue-specific stem cells Mesenchymal stem cells Induced pluripotent stem cells. Feng is working to generate iPS cells that may be more readily converted into dopaminergic neurons. Home Page. Search for topics, articles, videos, research, etc Hit enter to search or ESC to close.
Furthermore, owing to the pluripotency and self-renewal capacity, patient-specific iPSCs seem to be ideal cell source for the modeling of a rare genetic bone disease like osteopetrosis to identify osteoclast defects, leading to clinical heterogeneity in osteopetrosis patients, especially among those with different mutations in the same gene. Malignant infantile osteopetrosis MIOP is a rare genetic bone disease of childhood with no available treatment except allogeneic hematopoietic stem cell transplantations. Since MIOP is phenotypically and genotypically heterogeneous disease, there is still a great need for new research tools to identify the factors leading to defective osteoclastogenesis and its impact on bone metabolism and establishment of bone marrow hematopoietic niche.
Clinically relevant patient-specific iPSCs derived by integration-free reprogramming methods, like SeV and episomal vectors, will pave the way for disease modeling and discovery of new therapeutic targets, which will be of significant value to researchers and clinicians. The disease is classified into two types based on the mode of transmission: autosomal dominant osteopetrosis ADO and autosomal recessive osteopetrosis ARO [ 3 , 4 ].
A dysfunction of osteoclasts leads to bone resorption defects and increased bone density, decreased bone strength with abnormal bony overgrowth, and a bone marrow cavity insufficient to support hematopoiesis. Insufficient hematopoiesis results in bone marrow failure presented as severe cytopenias and compensatory extramedullary hematopoiesis.
Other clinical manifestations resulting from bony growth are cranial nerve dysfunctions, nasal obstruction, and gross motor developmental delays. Osteoclasts are derived from hematopoietic stem cells, thus the establishment of an allogeneic graft and differentiation of functional osteoclasts after transplantation help to alleviate bone remodeling and recovery from pancytopenia as well as extramedullary hematopoiesis.
Even though hematopoietic stem cell transplantation HSCT is the only curative treatment option, there is a high risk of graft failure and severe transplant-associated complications [ 1 ]. Generation of iPSCs is a milestone in stem cell research due to their unlimited maintenance capacity. Patient-specific iPSCs provide unique opportunities for both stem cell biologists and clinicians to dissect the pathogeneses and identify new alternative therapeutic strategies for rare genetic diseases by providing a virtually unlimited source of cells carrying the disease-causing mutations.
Gene delivery is crucial for the generation of iPSCs. To date, several alternatives have been developed and tested. Retroviral systems are characterized by high efficiency and reproducibility even in somatic cells that are difficult to reprogram. But they have some serious limitations such as stable expression of targeted transgenes, some of which are protooncogenes KLF4 and c-MYC and the possibility of insertional mutagenesis [ 8 , 9 ]. Therefore today, integration-free methods for the generation of iPSCs are in focus [ 10 , 11 ]. Somatic cell reprogramming can be achieved through various non-integrating methods such as mRNAs [ 12 , 13 ] or proteins [ 14 ] or gene transfer through vectors including Sendai virus SeV or episomal DNA vectors [ 15 ] while avoiding the risk of insertional mutagenesis [ 16 ].
However, reprogramming efficiency using either proteins or mRNAs is still very low, and these methods require repetitive delivery of multiple proteins or mRNAs into the somatic cells. Considering the need for reprogramming technologies with higher efficiency and better safety profile for translation into the clinic, episomal vectors and Sendai viral vector which both are non-integrative are accepted to be good alternatives [ 17 , 18 ].
Here, we used human bone marrow-derived mesenchymal stromal cells as the cell of origin, since they can be isolated and expanded easily from both donors and patients irrespective of their age, with a high proliferative potential. Also, they are multipotent progenitor cells, which may provide further reprogramming advantage considering the stemness of the parenteral cell [ 19 , 20 ].
This study aims to generate iPSCs from bone marrow-derived MSCs of osteopetrosis patients by using two different integration-free gene transfer methods episomal and Sendai viral vectors and compare the efficiency of two methods in order to select the most appropriate production method. The ultimate aim of this study is use patient-derived IPS cells as a research tool to investigate the factors leading to clinical heterogeneity as well as the discovery of modifier factors and genes in osteopetrosis patients, especially among those with different mutations in the same gene.
This might provide new insights into abnormal osteoclastogenesis and its role in the establishment of bone marrow hematopoietic stem cell niche in this rare genetic bone disease.
Induced Pluripotent Stem Cells (iPS) | UCLA Broad Stem Cell Center
Adipogenic and osteogenic differentiation potentials were evaluated for each established cell line. Cells were fixed and stained with Oil Red O stain to visualize fat droplets in the cells. Cells were fixed and stained with Alizarin red. The preparations of stained cells were visualized using an IX73 microscope Olympus. MSCs were reprogrammed by two different vector systems. One day prior to transduction, early passage P3-P4 MSCs were harvested with Accutase, plated in a 6-well plate coated with 0.
IPS colonies that started to appear around day 18 were harvested with manual microdissection method, transferred into Matrigel-coated dishes, and were expanded using EDTA clump passaging approach [ 23 ]. Following the identification of the IPS colonies, cells were harvested and maintained as described above.
The reprogramming efficiency was defined as the ratio of the number of colonies positive for alkaline phosphatase activity to the total number of cells used in the reprogramming experiment. Besides colony morphology and alkaline phosphatase activity, immunofluorescence staining, flow cytometry, and karyotype analyses were performed for the characterization of selected IPS lines.
Embryoid body formation and teratoma assays were performed to assess pluripotency in vitro and in vivo. As per each sample, three IPS lines were characterized. Gates on flow cytometry were defined according to isotype controls. Genetic stability of iPSC cell lines was evaluated by standard Giemsa-banded karyotype analysis.
Verification of the retained mutations was assessed by sequencing of the genomic locus. In vitro trilineage differentiation potential of iPSC lines was evaluated with embryoid body EB assay. Differentiated EBs were stained with hematoxylin and eosin. Microsections of paraffin-embedded EBs were also stained with antibodies against OCT4, vimentin, and synaptophysin. In vivo trilineage differentiation potential of iPSCs was demonstrated with teratoma assay.
Around 8-week post-injection terotoma formation was observed, mice were sacrificed and teratoma was dissected. Immunohistochemical stains for hematoxylin and eosin as well as Mallory trichrome and Toluidine Blue were performed for the evaluation of lineage-specific differentiation. Arbitrary gene expression levels were achieved by normalizing the gene of interest to the geometrical mean expressions of reference genes as described previously [ 24 ].
The arbitrary gene expression was further normalized to the mean expression of control samples to achieve fold change values. The data was analyzed using R statistical package version 3. BM-MSCs were characterized by morphology, immunophenotyping, and evaluation of differentiation potential. Mycoplasma testing proved that all MSC lines were free from contamination. We performed both SeV- and Epi5-mediated inductions from three patients who had disease-associated mutations in three different genes TCIRG1, SNX10, and CLCN7 representing the diverse genetic heterogeneity of osteopetrosis phenotype and two healthy donors, under the same culture conditions.
Induced Pluripotent Stem Cells for Regenerative Medicine
Three IPS lines per sample for each method were selected and characterized using the standard pluripotency assays. Reprogramming efficiencies were calculated and loss of SeV genome and Episomal plasmid were tested to understand the time-frame of the loss of episomal agents along with iPSC expansion. Finally, verification of the mutations was assured for all IPSC lines derived from osteopetrosis patients.
The first ES- like colonies were observed at around day 18 post-transduction with SeV and around day 21 post-transfection with episomal plasmids. Control plates were stained with alkaline phosphatase to identify iPSC colonies, and the mean efficiencies of successful reprogramming experiments of osteopetrotic BM-MSCs were found as 0.
Karyotype analyses were found normal for all iPSC lines Fig. Mutation verification showed that disease-related mutations were retained in the iPSCs. Immunostaining with pluripotency markers and DAPI. Relative expression of pluripotency genes in established iPSC lines.
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Relative arbitrary expression values are normalized to the negative control sample and indicated as fold change. Endogenous OCT4 and SOX2 expressions were analyzed differentially to demarcate any expression originating from the transgenes. The cumulative expressions were given for all the other genes. The analyses of pluripotency gene expression panel consisted of ten different target mRNA. Out of the four transgenes OSKM factors that were utilized for reprogramming, OCT4 and SOX2 were differentially assessed to delineate endogeneous versus exogenic transgene expression, since the timing and the levels of OCT4 and SOX2 expression are important indicators of reprogramming efficiency [ 25 ].
The pluripotency gene expressions were evaluated at different time points passages 5—20 for each established iPSC lines. Results revealed that the induction of pluripotency was already achieved at P5 and none of the investigated genes exhibited any significant time-course variance along the observed period for IPSC lines obtained with both methods.