Robust and highly efficient hiPSC generation from patient non-mobilized peripheral blood-derived CD34+ cells using the auto-erasable Sendai virus vector

Background: Disease modeling with patient-derived induced pluripotent stem cells (iPSCs) is a powerful tool forelucidating the mechanisms underlying disease pathogenesis and developing safe and effective treatments. Patientperipheral blood (PB) cells are used for iPSC generation in many cases since they can be collected with minimuminvasiveness. To derive iPSCs that lack immunoreceptor gene rearrangements, hematopoietic stem and progenitorcells (HSPCs) are often targeted as the reprogramming source. However, the current protocols generally requireHSPC mobilization and/or ex vivo expansion owing to their sparsity at the steady state and low reprogrammingefficiencies, making the overall procedure costly, laborious, and time-consuming.Methods: We have established a highly efficient method for generating iPSCs from non-mobilized PB-derivedCD34+ HSPCs. The source PB mononuclear cells were obtained from 1 healthy donor and 15 patients and werekept frozen until the scheduled iPSC generation. CD34+ HSPC enrichment was done using immunomagnetic beads,with no ex vivo expansion culture. To reprogram the CD34+-rich cells to pluripotency, the Sendai virus vectorSeVdp-302L was used to transfer four transcription factors: KLF4, OCT4, SOX2, and c-MYC. In this iPSC generationseries, the reprogramming efficiencies, success rates of iPSC line establishment, and progression time wererecorded. After generating the iPSC frozen stocks, the cell recovery and their residual transgenes, karyotypes, T cellreceptor gene rearrangement, pluripotency markers, and differentiation capability were examined.Results：We succeeded in establishing 223 iPSC lines with high reprogramming efficiencies from 15 patients with 8 different disease types. Our method allowed the rapid appearance of primary colonies (~ 8 days), all of which were expandable under feeder-free conditions, enabling robust establishment steps with less workload. After thawing, the established iPSC lines were verified to be pluripotency marker-positive and of non-T cell origin. A majority of the iPSC lines were confirmed to be transgene-free, with normal karyotypes. Their trilineage differentiation capability was also verified in a defined in vitro assay.Conclusion：This robust and highly efficient method enables the rapid and cost-effective establishment of transgene-free iPSC lines from a small volume of PB, thus facilitating the biobanking of patient-derived iPSCs and their use for the modeling of various diseases

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Comparisons of IC of inter-hemispheric attentional regions in the alpha band (8–13 Hz).

<p>(a) In left/right AG, the USN(+) group showed lower IC than the USN(-) group (uncorrected p < 0.05). (b) In left/right VAN, the USN(+) group showed lower IC than USN(-) (uncorrected p < 0.01). (c) In left/right DAN, no statistical difference was found among the groups.</p

Correlations between imaginary coherence and USN index in the alpha band (8–13 Hz).

<p>Scatter plot of data for 13 patients with brain damage. (a) A significant correlation with IC of the left/right AG and USN index was found (F(1,11) = 11.597, p = 0.006, r² = 0.513). (b) A correlation with IC of the left/right VAN and USN index showed a trend for significance (F(1,11) = 4.403, p = 0.058, r² = 0.286). (c) but no significance was found in the left/right DAN (F(1,11) = 1.442, p = 0.255, r² = 0.116). The solid line and dotted line represents regression line, 95% confidence intervals for the line of best fit, respectively.</p

Lesion overlap maps.

<p>USN(+) group (A) and USN(-) group (B). Horizontal sections through a template brain show the frequency of damage for each voxel. The color scale indicates the increasing frequency of overlapping lesions from violet (n = 1) to red (n = 6).</p

IC matrices among groups at each frequency band.

<p>The patterns of IC matrices in the delta (a) and theta band (b) were similar. The matrices in the beta band (d) and low-/high-gamma (e, f) band were also similar. Unlike the IC matrices in other frequency bands, the IC matrices in the alpha band (c) were considerably different among the groups. The color scale indicates imaginary coherence value (Z-transformed).</p

Illustration of IC matrix.

<p>The color scale indicates magnitude of imaginary coherence (Z-transformed). Area surrounded by green dashed line, and by pink dashed line indicates IC of left hemisphere and right hemisphere, respectively. Area surrounded by red dashed line and by blue dashed line indicates IC of the DAN and the VAN, respectively. Yellowed areas indicate inter-hemispherical homologous regions. SFG: superior frontal gyrus; SPL: superior parietal lobule; MT: middle temporal region; VFG: ventral frontal gyrus; IFG: inferior frontal gyrus; SMG: supramarginal gyrus; AG: angular gyrus; STG: superior temporal gyrus.</p