Generation of iPSC lines with high cytogenetic stability from peripheral blood mononuclear cells (PBMCs)

Panther, Lindsay; Ornelas, Loren; Jones, Michelle R.; Gross, Andrew; Gomez, Emilda; Liu, Chunyan; Berman, Benjamin P.; Svendsen, Clive N.; Sareen, Dhruv

doi:10.1101/2021.09.27.462082

Cited by 4 publications

(6 citation statements)

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“…Reprogramming PBMCs into iPSCs was based on methods that favored both lymphoid T cells and myeloid non-T cells. 7 , 9 We initially attempted to avoid T cells; however, reprogramming non-T cells was less efficient, and hence, using solely non-T cells was not feasible for iPSC production at scale. As such, iPSC lines in the Answer ALS cohort were derived from both T cells and non-T cells ( Figure 2A ).…”

Section: Resultsmentioning

confidence: 99%

“…Many iPSC lines in the ALS disease modeling community have been fibroblast-derived. However, the improved cytogenetic stability of PBMC-derived versus fibroblast-derived iPSCs, 9 combined with their ease of collection and lack of expansion requirement, make PBMCs a preferable source of iPSCs at scale. Additionally, a patient’s genetics typically override the differences caused by a somatic cell of origin, and differentiation propensities are not suspected to be influenced by whether the iPSC line was fibroblast or blood-derived 30 suggesting that the PBMC source of iPSCs in this dataset should not confound comparisons with other datasets using fibroblast-derived iPSCs.…”

Section: Discussionmentioning

confidence: 99%

“…Patient enrollment, clinical data collection, blood sampling, and reprogramming and QC of iPSC lines has been described previously. 5 , 7 , 9 Briefly, 5×10 6 PBMCs isolated from the buffy coat of patient blood samples were nucleofected with plasmids pEP4 E02S ET2K, 46 pCXLE-hOCT3/4-shp53-F, 47 pCXLE-hUL, 47 pCXLE-hSK, 47 and pCXWB-EBNA1 48 using the Amaxa Human T cell Nucleofector Kit and Nucleofector 2D Device. Following nucleofection, cells were resuspended in either T cell media (X-VIVO 10 media supplemented with 30U/mL IL-2 and 5 μl/well Dynabeads Human T-activator CD3/CD28) or non-T cell medium (αMEM supplemented with 10%heat inactivated FBS, 10 ng/ml IL-3, 10 ng/ml IL-6, 10 ng/ml G-CSF, and 10 ng/ml GM-CSF), plated onto mitomycin treated mouse embryonic fibroblasts, and placed in a 37°C incubator.…”

Section: Methodsmentioning

confidence: 99%

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Large-scale differentiation of iPSC-derived motor neurons from ALS and control subjects

Workman¹,

Lim²,

Wu³

et al. 2023

Neuron

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

Large-scale differentiation of iPSC-derived motor neurons from ALS and control subjects

Workman¹,

Lim²,

Wu³

et al. 2023

Neuron

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“…Additionally, EPCs are frequently replenished in the blood and therefore are less likely to accumulate environment-induced mutations like fibroblasts (Panther et al, 2021; Kamimura et al, 2021). Moreover, they lack the TCR/BCR genes recombination found in T-cells, making them a more attractive source of iPS cells.…”

Section: Discussionmentioning

confidence: 99%

HLA-Based Banking of Human Induced Pluripotent Stem Cells in Saudi Arabia

Alowaysi,

Lehmann,

Al-Shehri

et al. 2023

Preprint

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Human iPSCs' derivation and use in clinical studies are transforming medicine. Yet, there is a high cost and long waiting time for autologous iPS-based cellular therapy, and the genetic engineering of hypo-immunogenic iPS cell lines is hampered with numerous hurdles. Therefore, it is increasingly interesting to create cell stocks based on HLA haplotype distribution in a given population. In this study, we assessed the potential of HLA-based iPS banking for the Saudi population. First, we analyzed the HLA database of the Saudi Stem Cell Donor Registry (SSCDR), which contains high-resolution HLA genotype data of 64,315 registered Saudi donors at the time of analysis. We found that only 13 iPS lines would be required to cover 30% of the Saudi population, 39 iPS lines would offer 50% coverage and 596 for more than 90% coverage. Next, As a proof-of-concept, we launched the first HLA-based banking of iPSCs in Saudi Arabia. Using clinically relevant methods, we generated the first iPSC line from a homozygous donor for the most common HLA haplotype in Saudi. The two generated clones expressed pluripotency markers, could be differentiated into all three germ layers, beating cardiomyocytes and neuronal progenitors. To ensure that our reprogramming method generates genetically stable iPSCs, we assessed the mutational burden in the generated clones and the original blood sample from which the iPSCs were derived using whole-genome sequencing. All detected variants were found in the original donor sample and were classified as benign according to current guidelines of the American College of Medical Genetics and Genomics (ACMG). This study sets a road map for introducing iPS-based cell therapy in the Kingdom of Saudi Arabia.

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“…Additionally, EPCs are frequently replenished in the blood and therefore are less likely to accumulate environment-induced mutations like fibroblasts [ 33 , 34 ]. Moreover, they lack the TCR/BCR genes recombination found in T-cells, making them a more attractive source of iPS cells.…”

Section: Discussionmentioning

confidence: 99%

HLA-based banking of induced pluripotent stem cells in Saudi Arabia

Alowaysi,

Lehmann,

Al-Shehri

et al. 2023

Stem Cell Res Ther

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Background Human iPSCs' derivation and use in clinical studies are transforming medicine. Yet, there is a high cost and long waiting time associated with autologous iPS-based cellular therapy, and the genetic engineering of hypo-immunogenic iPS cell lines is hampered with numerous hurdles. Therefore, it is increasingly interesting to create cell stocks based on HLA haplotype distribution in a given population. This study aimed to assess the potential of HLA-based iPS banking for the Saudi population. Methods In this study, we interrogated the HLA database of the Saudi Stem Cell Donor Registry (SSCDR), containing high-resolution HLA genotype data from 64,315 registered Saudi donors at the time of analysis. This database was considered to be a representative sample of the Saudi population. The most frequent HLA haplotypes in the Saudi population were determined, and an in-house developed iterative algorithm was used to identify their HLA matching percentages in the SSCDR database and cumulative coverage. Subsequently, to develop a clinically relevant protocol for iPSCs generation, and to illustrate the applicability of the concept of HLA-based banking for cell therapy purposes, the first HLA-based iPS cell line in Saudi Arabia was generated. Clinically relevant methods were employed to generate the two iPS clones from a homozygous donor for the most prevalent HLA haplotype in the Saudi population. The generated lines were then assessed for pluripotency markers, and their ability to differentiate into all three germ layers, beating cardiomyocytes, and neural progenitors was examined. Additionally, the genetic stability of the HLA-iPS cell lines was verified by comparing the mutational burden in the clones and the original blood sample, using whole-genome sequencing. The standards set by the American College of Medical Genetics and Genomics (ACMG) were used to determine the clinical significance of identified variants. Results The analysis revealed that the establishment of only 13 iPSC lines would match 30% of the Saudi population, 39 lines would attain 50% coverage, and 596 lines would be necessary for over 90% coverage. The proof-of-concept HLA-iPSCs, which cover 6.1% of the Saudi population, successfully demonstrated pluripotency and the ability to differentiate into various cell types including beating cardiomyocytes and neuronal progenitors. The comprehensive genetic analysis corroborated that all identified variants in the derived iPSCs were inherently present in the original donor sample and were classified as benign according to the standards set by the ACMG. Conclusions Our study sets a road map for introducing iPS-based cell therapy in the Kingdom of Saudi Arabia. It underscores the pragmatic approach of HLA-based iPSC banking which circumvents the limitations of autologous iPS-based cellular therapies. The successful generation and validation of iPSC lines based on the most prevalent HLA haplotype in the Saudi population signify a promising step toward broadening the accessibility and applicability of stem cell therapies and regenerative medicine in Saudi Arabia.

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Generation of iPSC lines with high cytogenetic stability from peripheral blood mononuclear cells (PBMCs)

Cited by 4 publications

References 100 publications

Large-scale differentiation of iPSC-derived motor neurons from ALS and control subjects

Large-scale differentiation of iPSC-derived motor neurons from ALS and control subjects

HLA-Based Banking of Human Induced Pluripotent Stem Cells in Saudi Arabia

HLA-based banking of induced pluripotent stem cells in Saudi Arabia

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