Method for improving efficiency of reprogramming PBMC into iPSC

Abstract

The method for improving the efficiency of reprogramming the PBMC into the iPSC mainly comprises the following culture steps: separating out the PBMC from freshly collected peripheral blood, culturing the PBMC in a culture medium added with multicellular factors UM171, SCF, ANGPTL3, TPO, Flt3L, IL-3, IL-6 and TFF3 so as to improve the proportion of CD34, then carrying out electroporation reprogramming, and optimizing cell culture conditions after reprogramming to obtain the iPS. According to the method, the multi-cell heterozygosis characteristic of the PBMC is fully exerted, the proportion of CD34 cells is effectively increased while the PBMC environment stability is maintained by applying a technical means, more original cells are provided for reprogramming of the iPSC, and the efficiency of reprogramming the PBMC into the iPSC is improved under the assistance of a more stable post-electrotransformation culture environment.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a method for improving the efficiency of reprogramming peripheral blood mononuclear cells (PBMCs) into iPSCs. Background Technology

[0002] Regenerative medicine, as a cutting-edge technological field, is reshaping the modern medical landscape with its unique therapeutic potential. Breakthroughs in cell therapy, especially in stem cell research, have provided new pathways to overcome diseases that are difficult to address with traditional medicine. Induced pluripotent stem cells (iPSCs) are pluripotent stem cells obtained through somatic cell reprogramming technology. Essentially, they involve inducing differentiated somatic cells to revert to their original pluripotent state using specific reprogramming factors. iPSCs possess theoretically unlimited proliferative capacity and the ability to differentiate into cells from the three germ layers (endoderm, mesoderm, and ectoderm), thus showing broad application prospects in regenerative medicine, disease model construction, and drug development. Compared to embryonic stem cells (ESCs), iPSCs are derived without ethical controversies and avoid the complexity and high costs of traditional embryonic stem cell acquisition, making them an important technological platform for current life science research and clinical applications.

[0003] In iPSC reprogramming technology, traditional methods typically use skin fibroblasts as source cells. However, this method requires surgical tissue sampling, and the invasive procedure causes trauma to the donor, limiting its clinical application. In contrast, peripheral blood mononuclear cells (PBMCs) can be directly isolated from peripheral blood, offering advantages such as convenient sampling, non-invasiveness, and high reproducibility, making them easier to promote in practical applications. Despite the significant advantage of PBMCs in sampling convenience, their reprogramming efficiency is generally low, mainly due to insufficient stability of PBMC quality and their highly enclosed state as terminally differentiated cells. To address the low reprogramming efficiency of PBMCs, existing reprogramming solutions include direct transfection with Yamanaka factors (Oct4, Sox2, Klf4, and c-Myc, abbreviated as OKSM), introducing in vitro transcribed mRNA into cells using non-viral methods, using non-integrating plasmids, and Sendai virus infection. However, even with these different technical approaches, the reprogramming efficiency of PBMCs typically remains at a low level of 0.01%-1%. Studies suggest this phenomenon may be related to the unique gene expression profile and epigenetic modifications of PBMCs, leading to higher technical barriers during reprogramming. Notably, CD34⁺ cells, as hematopoietic progenitor cells, exhibit higher reprogramming efficiency within the PBMC population. Compared to other PBMC subpopulations, CD34⁺ cells possess more primitive cellular characteristics, with their gene expression profile and epigenetic state more closely resembling those of pluripotent stem cells. Therefore, specifically increasing the proportion of CD34⁺ cells in PBMCs can effectively improve reprogramming efficiency. This finding provides an important theoretical basis for optimizing PBMC reprogramming conditions. Furthermore, the influence of culture conditions on PBMC reprogramming efficiency is equally significant. Summary of the Invention

[0004] Based on existing technologies, this invention aims to propose a novel PBMC reprogramming culture system. By optimizing cell isolation and culture environment, specifically by synergistically improving the reprogramming efficiency of PBMCs to iPSCs from three dimensions: cell pretreatment, culture environment regulation, and culture medium optimization, this system also takes into account the operability and cost-effectiveness of the technology. This provides a more efficient and stable solution for the application of iPSCs in regenerative medicine, disease model construction, and drug development.

[0005] To achieve the objective of this invention, the present invention provides a method for improving the efficiency of peripheral blood mononuclear cells (PBMCs) reprogrammed into induced pluripotent stem cells (iPSCs), characterized by the following steps of culturing the cells under hypoxic conditions of 1–3% O2 ​​and 5% CO2 and normoxic conditions of 20–22% O2 and 5% CO2: S1. PBMC pretreatment and CD34⁺ cell expansion culture: PBMCs were isolated from freshly collected peripheral blood and seeded into non-tissue culture dishes. CD34⁺ cells were expanded using serum-free PBMC expansion medium supplemented with multiple cytokines UM171, SCF, ANGPTL3, TPO, Flt3L, IL-3, IL-6 and TFF3. After 4 days of culture, the proportion of CD34⁺ cells was ≥20%. The first 48 hours were in a hypoxic environment and the next 48 hours were in a normoxic environment. 50% of the culture medium was replaced every 24 hours. S2. Efficient PBMC Reprogramming and iPSC Cloning Induction: PBMCs pre-expanded with CD34⁺ cells cultured in step S1 were electrotransferred with reprogramming factors; after electrotransfer... PBMC Cells were seeded in glassnein (VTN)-coated culture dishes and then reprogrammed through staged medium changes: PBMC electroporated cells were adapted using PBMC amplification medium supplemented with 0.01–0.05 mM α-tocopherol; PBMC electroporated cells were induced to undergo N2B27 induction culture using N2B27 electroporation reprogramming medium, with sodium butyrate, PS48, and IHMT-MST1-39 added to the medium for reprogramming induction; and PBMC electroporated cells were induced to become iPSCs using complete iPSC medium containing Y27632, PD0325901, CHIR99021, SB431542, and Hetrombopag, ultimately obtaining iPSC clones.

[0006] Furthermore, in step S1, the source of the PBMCs needs to be controlled. The peripheral blood is separated into PBMCs by Ficoll density gradient centrifugation. The time from peripheral blood collection to the completion of PBMC separation does not exceed 4 hours, and the blood is kept at 2-8°C throughout the process to maintain cell biological activity.

[0007] The serum-free PBMC amplification medium used in step S1 is X-VIVO 15 basal medium. The cytokines added to the medium and the amounts added are as follows: 15-25 ng / mL UM171, 45-55 ng / mL SCF, 95-105 ng / mL ANGPTL3, 5-15 ng / mL TPO, 45-55 ng / mL Flt3L, 15-25 ng / mL IL-3, 15-25 ng / mL IL-6, and 50-100 ng / mL TFF3.

[0008] In step S1, the PBMCs obtained after peripheral blood separation are processed at a concentration of 5 × 10⁻⁶. 4 ~5×10 5The cells were seeded at a density of cells / mL onto the culture surface of non-tissue culture vessels to avoid excessive cell aggregation and apoptosis after culture.

[0009] In step S2, the electrically transferred PBMC operates at a speed of 1 to 2 × 10⁻⁶. 5 Cells / wells were seeded at a density of 1.5–2 μg / cm² in 6-well plates coated with quinone. 2 .

[0010] Furthermore, in step S2, the process of reprogramming induced in PBMCs after electroporation through phased culture medium conversion is as follows: (1) Adaptive culture of PBMC electroporated cells: On the same day after electroporation, PBMCs were cultured in normoxic conditions for 24 hours in PBMC amplification medium supplemented with 0.01~0.05mM α-tocopherol. (2) N2B27-induced culture of PBMC electroporated cells: On the day after electroporation, half of the culture medium was replaced with N2B27 electroporation reprogramming medium supplemented with 0.2-0.3mM sodium butyrate, 5-10 μM PS48 and 1-3 μM IHMT-MST1-39, and the cells were cultured under normoxic conditions; On the 3rd and 4th days of electroporation, half of the culture medium was replaced with N2B27 electroporation reprogramming medium supplemented with 0.2-0.3mM sodium butyrate and 5-10 μM PS48 every 24 hours; Starting from the 5th day of electroporation, half of the culture medium was replaced with N2B27 electroporation reprogramming medium supplemented with 5-10 μM PS48 every 24 hours; From the 3rd to the 5th day of electroporation, hypoxia-induced culture was carried out in a hypoxic environment for 2-3 days, and thereafter the cells were cultured under normoxic conditions. (3) PBMC electroporation to induce iPSC culture: On days 9-10 of electroporation, replace 50% of the N2B27 reprogramming medium with iPSC complete medium. Within 72 hours after the medium change, replenish iPSC complete medium by replacing half of the medium every 24 hours. In the next 24-48 hours, replenish iPSC complete medium by replacing 2 / 3 of the medium every 24 hours. After that, replace all the iPSC complete medium every 48 hours.

[0011] The iPSC complete culture medium contains 9–14 μM Y27632, 0.5–1 μM PD0325901, 5–10 μM CHIR99021, 2–5 μM SB431542, and 5–10 μM Hetrombopag; and after iPS cells appear during the culture process, the concentration of Y27632 in the replaced iPSC complete culture medium is adjusted to 10 μM.

[0012] Further, in step S2, the electrotransfer of reprogramming factors to PBMCs is performed using a Neon electroporator. In a buffer containing 10% Ficoll PM400, a non-integrative Epi5 attachor vector pre-loaded with the OCT4, SOX2, KLF4, c-MYC, mp53DD, and EBNA1 genes is electrotransferred into the PBMCs to complete the plasmid transfection for reprogramming. The electrotransfer parameters are set to 1650V, 10ms, and 3 pulses. The reprogramming protocol used is the electrotransfer of the non-integrative Epi5 attachor vector into the PBMCs to complete the plasmid transfection for reprogramming.

[0013] In step S2, the electrotransfer of reprogramming factors to PBMCs is performed after the PBMCs have been expanded and cultured with CD34⁺ cells and the proportion of CD34⁺ cells has reached the target. The electrotransferred PBMCs need to be immediately transferred to a culture dish coated with porphyrin and supplemented with the corresponding PBMC expansion medium for a 24-hour adaptive culture to maintain the viability of the electrotransferred cells.

[0014] In step S2, the culture vessel coated with hydrin is a 6-well plate coated with hydrin, and the hydrin coating concentration is 1.5–2 μg / cm³. 2 .

[0015] In step S2, the PBMC amplification medium used for the PBMC electroporation cell adaptation culture is a PBMC-USATF36 amplification medium supplemented with 0.01~0.05mM α-tocopherol.

[0016] The beneficial effects of this invention are as follows: Step S1 of this invention provides a novel PBMC pretreatment culture protocol to improve cell quality and better complete the reprogramming task. In step S1, the PBMC culture protocol uses serum-free PBMC amplification medium, preferably X-VIVO 15 basal medium, and adds the undetermined cytokines UM171, SCF, Flt3L, TPO, ANGPTL3, IL-3, IL-6 and TFF3. After culture, CD34⁺ cells in PBMCs are efficiently amplified. The proportion of CD34⁺ cells is detected by flow cytometry. The proportion of CD34 positive cells is ≥20%, which is a 10-15 fold increase compared to the initial proportion of CD34⁺ cells in PBMCs (usually ≤2.5%).

[0017] Step S2 of this invention provides a highly efficient reprogramming and iPSC cloning induction culture scheme for PBMCs. Firstly, it employs a dynamic oxygen regulation strategy: after electroporation, PBMCs are induced to undergo hypoxia in a low-oxygen (1%-3%) environment for 2-3 days during the early stages of culture; at other times during the culture period, cells are cultured under normoxic conditions. Secondly, it provides a time-controlled reprogramming culture system: through the synergistic optimization of staged culture and environmental parameters, the reprogramming efficiency of PBMCs to iPSCs is significantly improved.

[0018] In step S2, the electroporated PBMCs were transferred to a glassnein-coated culture dish and cultured for 24 hours in PBMC amplification medium supplemented with α-tocopherol to maintain cell viability. The day after electroporation, half the volume of the PBMC amplification medium was replaced with N2B27 electroporation reprogramming medium (supplemented with 0.2–0.3 mM sodium butyrate and 5–10 μM sodium butyrate). Cells were cultured in N2B27 electroporation medium supplemented with 0.2-0.3 mM sodium butyrate and 5-10 μM PS48 (PS48) for reprogramming induction. On days 3 and 4 after electroporation, half of the medium was replaced every 24 hours with N2B27 medium supplemented with 0.2-0.3 mM sodium butyrate and 5-10 μM PS48 to increase reprogramming efficiency. From day 5 onwards, N2B27 medium supplemented with 5-10 μM PS48 was used for induction culture. During the early stages of culture (days 3-5), cells were induced to undergo hypoxia under a low-oxygen environment to accelerate the reprogramming process, after which the oxygen concentration was restored to normoxic conditions for normal culture. After N2B27 induction culture, the cells entered the iPSC induction culture stage, with iPSC complete medium gradually added to promote the formation of more iPSC clones.

[0019] In summary, this invention, through optimizing PBMC culture conditions, adjusting the culture system and environment changes after electroporation, and combining multiple culture media for sequential culture, significantly improves PBMC reprogramming efficiency, thereby achieving more efficient iPSC preparation. Simultaneously, it reduces operational complexity and cost, providing a more efficient and stable solution for iPSC applications in regenerative medicine, disease model construction, and drug development. Furthermore, the solution provided by this invention is based on a serum-free culture protocol, effectively reducing uncontrollable factors such as pathogen contamination and batch-to-batch variations caused by exogenous serum, achieving safe, effective, and easily reproducible reprogramming efficiency improvements.

[0020] To better understand the invention and more clearly describe the various steps and operation schemes of the invention, the technical solution of the invention will be described in detail below with reference to the accompanying drawings and embodiments. Attached Figure Description

[0021] Figure 1 This is a schematic diagram of the basic operation process of reprogramming a PBMC into an iPSC according to the present invention; Figure 2 Comparison of CD34⁺ cell expansion efficiency between cryopreserved and fresh PBMCs cultured using the method of this invention and conventional culture. The markings in the figure are explained as follows: 2a -- The proportion of CD34⁺ in frozen PBMCs; 2b -- The proportion of CD34⁺ in cryopreserved PBMCs after culturing in a conventional culture system; 2c -- The proportion of CD34⁺ in frozen PBMCs after culturing using the culture system of this invention; 2d -- The proportion of CD34⁺ in fresh PBMCs; 2e -- Fresh PBMCs, CD34⁺ ratio after being cultured in a conventional culture system; 2f -- Fresh PBMCs, the proportion of CD34⁺ after being cultured using the culture system of this invention; Figure 3 This is a comparison chart of the adaptation states of PBMCs under different culture conditions via electroporation reprogramming. The markings in the chart are explained below: 3a -- Post-culture state of PBMCs in a traditional culture system; 3b -- The state of PBMCs after culture in the culture system of this invention; 3c -- Cell state the day after electroporation in a traditional culture system; 3d -- Cell state after adaptive culture the day after electroporation in the culture system of this invention; 3e--In the traditional culture system, on the 10th day after electroporation, the cells were basically in suspension. 3f--In the culture system of this invention, on the 10th day after electroporation, the cells showed a state of adaptation to the stem cell culture environment, the cells became longer and no longer round, and had a tendency to adhere to the wall; Figure 4 This is a comparison chart of the iPSC clone formation process, with the markings explained below: 4a--In the traditional culture system, some adherent iPS-like cells appeared on day 18 after electroporation; 4b—In the culture system of this invention, adherent iPS-like cells begin to appear on day 15 after electroporation; 4c -- Under the traditional culture system, relatively obvious ipsc sample colonies appeared on day 26 after electroporation; 4d -- In the culture system of this invention, on the 22nd day after electroporation, obvious iPSC clonal clusters of cells appeared; 4e -- Under the traditional culture system, a large iPSC clone structure appeared on day 33 after electroporation; 4f--In the culture system of this invention, on the 28th day after electroporation, the iPSC clonal cluster structure is obvious and presents a uniform undifferentiated clonal morphology; 4g -- The state of iPSC clones cultured in the culture system of this invention after being picked and cultured for 5 days for identification; Figure 5 This is an immunofluorescence staining image of the pluripotency markers of iPSC clones cultured in the culture system of this invention; Figure 6 This is a comparison chart of the efficiency of traditional culture and the culture system of this invention. Detailed Implementation

[0022] The present invention will now be described in further detail with reference to the accompanying drawings, specific embodiments, and comparative examples.

[0023] Experimental conditions and reagent descriptions: Unless otherwise specified, experimental methods in the embodiments of this invention were performed under conventional conditions or as recommended by the manufacturer. All commonly used chemical reagents used in the embodiments are commercially available products to ensure the standardization and repeatability of experimental conditions. Example 1

[0024] This embodiment follows Figure 1 The PBMC-iPSC induction and reprogramming process shown demonstrates the induction of iPSCs. Through the establishment of an innovative step-by-step culture system, the entire process from cell source control, pretreatment and expansion, reprogramming induction to the establishment of pluripotent stem cells is optimized, significantly improving the efficiency of reprogramming and the quality of the final cell products.

[0025] This invention provides a method for improving the efficiency of reprogramming PBMC to iPSC, comprising two interconnected and synergistic core stages: Phase 1: PBMC Pretreatment and High-Efficiency CD34⁺ Cell Expansion. This phase aims to obtain a large number of starter cells suitable for reprogramming. First, the cell source is strictly controlled, using fresh peripheral blood anticoagulated with heparin sodium (collection to separation time ≤ 4 hours, stored at 2-8℃ throughout). PBMCs are separated by Ficoll-Paque PLUS density gradient centrifugation and thoroughly washed with DPBS. Subsequently, PBMCs are centrifuged at 3 × 10⁶ cells / day. 5 Cells / mL were seeded onto non-tissue-treated culture surfaces and cultured using a dedicated serum-free amplification medium. This medium was based on X-VIVO-15 and supplemented with an optimized combination of cytokines, including: 20±5 ng / mL UM171, 50±5 ng / mL SCF, 100±5 ng / mL ANGPTL3, 10±5 ng / mL TPO, 50±5 ng / mL Flt3L, 20±5 ng / mL IL-3, 20±5 ng / mL IL-6, and 75±15 ng / mL TFF3. A unique time-series oxygen concentration control was employed: the first 48 hours were conducted under a hypoxic environment of 3% O2 ​​and 5% CO2 to promote the proliferation and survival of hematopoietic stem / progenitor cells; the following 48 hours were converted to a normoxic environment of 21% O2 and 5% CO2 to prepare for subsequent reprogramming. 50% of the medium was replaced every 24 hours during this period. After this 4-day pretreatment, the proportion of CD34⁺ cells was significantly increased to ≥20% by flow cytometry (anti-human CD34-APC antibody), achieving an amplification of more than 12 times compared to the initial state, laying a solid foundation for reprogramming.

[0026] Phase Two: Efficient Reprogramming and iPSC Cloning Induction. This phase is crucial for iPSC generation. First, take 1×10 5Pre-amplified cells were electroporated using the Neon electroporation system in a buffer containing 10% Ficoll PM400 with a non-integrated Epi5 episome vector preloaded with the genes OCT4, SOX2, KLF4, c-MYC, mp53DD, and EBNA1. The electroporation parameters were set to 1650V, 10ms, and 3 pulses. Immediately after electroporation, cells were transferred to porphyrin-coated culture plates (coating concentration 1.75 ± 0.25 μg / cm²). To alleviate cellular stress from electroporation, cells were initially cultured for 24 hours in PBMC amplification medium supplemented with 0.03 mM α-tocopherol under normoxic (21% O2) conditions for acclimatization. Subsequently, a culture medium time-series conversion procedure was initiated: On the following day, the medium was replaced with a mixture of 50% N2B27 electroporation reprogramming medium and 50% PBMC amplification medium, with the addition of 0.25 mM sodium butyrate, 5 μM PS48, and 2 μM IHMT-MST1-39; from day 3 onwards, the medium was completely converted to N2B27 reprogramming medium, with the addition of 0.25 mM sodium butyrate (continued until day 4) and 5 μM PS48 (continued until day 9) to synergistically promote chromatin remodeling and activate reprogramming-related signaling pathways. Crucially, from day 3 to day 5, cells were cultured in a hypoxic environment of 3% O2, which significantly reduced reactive oxygen species (ROS) levels and mitigated cell damage. Around day 9, when cells begin to adhere and exhibit morphological changes, the culture medium is gradually replaced with a modified StemFlex complete medium specifically for iPSC colony formation. This medium, in addition to the basic components, is supplemented with 10 μM MY27632, 0.5 μM PD0325901, 5 μM CHIR99021, 2 μM SB431542, and 5 μM Hetrombopag to establish and stabilize the pluripotent network. After complete replacement, a complete medium change is performed every 48 hours until typical iPSC colonies with well-defined borders, large nuclei, and sparse cytoplasm appear.

[0027] The systematic processing described above yielded iPSC clones with typical morphology, dense structure, and good pluripotency. The entire process utilizes a serum-free culture system with clearly defined components, fundamentally eliminating the risk of pathogen contamination and batch-to-batch variability from exogenous serum, resulting in high reproducibility and stability. This method is not only relatively simple to operate and cost-effective, but also provides an efficient, stable, and safe cell source for iPSC-based regenerative medicine research, disease model construction, and drug screening, demonstrating broad prospects for industrial application.

[0028] To further illustrate the superiority of the technical solution of the present invention, the solution of the present invention will be implemented step by step below, and a comparative example will be set up to compare and illustrate the experimental results.

[0029] Example 2: Isolation and Culture Scheme of PBMCs of the Present Invention (a) PBMC separation Fresh (taken <4h) human peripheral blood was used as the starting material for inducing reprogramming. PBMCs were isolated and prepared using PBMC separation reagent, Ficoll density gradient centrifugation, and following the separation protocol provided by the reagent supplier. After washing with DPBS, the PBMCs were collected in 50mL centrifuge tubes and entered the PBMC culture stage.

[0030] (II) Cultivation of PBMCs Culture medium preparation: Prepare PBMC amplification medium up to 1 day in advance, using the following method: Based on X-VIVO-15, add an optimized combination of cytokines, including: 20±5 ng / mL UM171, 50±5 ng / mL SCF, 100±5 ng / mL ANGPTL3, 10±5 ng / mL TPO, 50±5 ng / mL Flt3L, 20±5 ng / mL IL-3, 20±5 ng / mL IL-6, and 75±15 ng / mL TFF3. During preparation, accurately calculate and add each cytokine according to the actual consumption of the medium to ensure stable cytokine concentrations. After preparation, immediately sterilize by filtration through a 0.22 μm filter and aliquot into 50 mL tubes. Store at 4°C and use within 7 days. Before each use, preheat the medium in a 37°C water bath to ensure the cells are cultured at a suitable temperature.

[0031] Hypoxia-induced culture: The isolated PBMCs were resuspended in preheated PBMC amplification medium, and the cell density was adjusted to 3 × 10⁶ cells / year. 5 Cells / mL. Seed the cell suspension into untreated 10 cm culture dishes, adding an appropriate amount of preheated PBMC amplification medium to each dish, ensuring even coverage. Immediately place the dishes in hypoxic conditions (1–3% O2, 5% CO2, 37°C) for induction culture for 48 hours. During this period, after 24 hours of culture, perform a half-volume medium replacement: discard half of the old medium and add an equal volume of fresh, preheated PBMC amplification medium to maintain the stability of the culture environment and cell viability.

[0032] Normative culture: After culturing continuously under hypoxic conditions for 48 hours, the culture dishes were removed, half of the old culture medium was discarded, and the medium was replaced with an equal volume of preheated PBMC amplification medium. The culture dishes were then transferred to a normoxic incubator (21% O2, 5% CO2, 37℃) for another 48 hours. During this stage, half of the culture medium was replaced every 24 hours to ensure cell growth in a suitable environment.

[0033] Culture success verification: After 4 days of conditional culture, PBMCs undergo rigorous cell counting and CD34 positivity analysis to verify the success of the induced culture. Flow cytometry is used; a CD34⁺ cell percentage ≥ 20% is required to proceed to the next stage. (See attached diagram) Figure 2 As shown in Figure 2f. During the CD34 positive verification process, PBMCs were kept under suitable culture conditions and environment to maintain cell viability and stability, facilitating subsequent experiments.

[0034] Example 3: Electrical reprogramming stage of the PBMC in this invention 3.1 Preparation of culture medium: To ensure stable cell attachment and growth after electroporation, the culture medium must be prepared in advance. Coat 6-well plates with VTN, ensuring a final planar coating concentration greater than 1.5 μg / cm². Add VTN solution to each well, ensuring complete coverage of the bottom. Incubate the coated 6-well plates at 37°C for at least 1 hour to ensure sufficient VTN adsorption. After coating, discard excess VTN solution and wash twice with DPBS to remove any unadsorbed VTN. Then, add 2 mL of preheated PBMC amplification medium to each well, supplement with an additional 0.03 mM α-tocopherol, and incubate at 37°C for use within 4 hours.

[0035] 3.2 Preparation for electroporation: like Figure 3 As shown in section 3.b, before electroporation, successfully induced PBMCs need to be processed to ensure consistency in cell morphology and quality. The specific steps are as follows: Collect PBMCs induced in Example 2 and verified by flow cytometry to have a CD34⁺ ratio ≥20%, wash twice with DPBS (centrifugation conditions: 300g, 10 min, room temperature) to thoroughly remove residual culture medium components. Accurately count the cells using the trypan blue exclusion method or an automated cell counter, and adjust the cell concentration to 1×10⁻⁶. 5 Cells / 10μL (using dedicated electroporation buffer, such as Neon® Electrolytic Buffer). Transfer the cell suspension to a 1.5 mL centrifuge tube, centrifuge at 300g for 10 minutes, and thoroughly aspirate the supernatant using 200 μL and 10 μL pipettes to ensure no liquid residue remains.

[0036] 3.3 Cell electroporation: Electroporation must be performed strictly according to the instruction manual of the electroporation instrument to ensure standardized and safe experimental conditions. The specific steps are as follows: Thaw the non-integrative plasmid vector and other relevant reagents in a 37°C water bath, then immediately transfer them to ice for storage to ensure plasmid stability and activity. Add an appropriate amount of buffer and plasmid vector to an EP tube (after completely discarding DPBS). The specific amounts should be adjusted according to the reaction system and cell volume.

[0037] For the Neon NxT electroporation system, the operating procedures are as follows: Place a tube containing 2 mL of electroporation buffer in the tube electroporation station. Use the electroporation tip to aspirate the prepared plasmid-cell suspension and transfer it into the tube. Set the electroporation parameters according to the instrument manual: Pulse voltage 1650 V, Pulse width 10 ms, Pulse number 3, Tip type 10 μL. Perform the electroporation operation, ensuring the parameters are set accurately. After electroporation is complete, immediately remove the cells from the electroporation chamber.

[0038] Example 4: Reprogramming the cell adaptation induction (early stage) culture stage according to the present invention 4.1 Adaptation culture phase on the day of electroporation: Cells electroporated in Example 3 were immediately seeded into VTN-coated plates containing PBMC amplification medium supplemented with an additional 0.03 mM α-tocopherol, pre-equilibrated in step 3.1, to establish a suitable culture environment. The culture plates were placed in an incubator at 37°C, 5% CO2, and normoxic (21% O2) for 24 hours for electroporation adaptation. This stage aims to maintain the basic state and viability of the cells, avoiding cellular stress caused by the electroporation operation. It also aims to alleviate membrane potential stress and osmotic pressure shock caused by the electroporation operation, providing a stable microenvironment for the expression of exogenous reprogramming factors. During this period, no interference was performed on the cells to ensure they recovered and adjusted in a stable environment. The cells gradually adapted to the new culture environment during this stage, laying the foundation for the subsequent reprogramming process.

[0039] 4.2 Adaptive conversion of reprogramming conditions after electrical transfer: The day after electroporation, to promote further cell adaptation and the expression of reprogramming factors, the culture medium needs to be adjusted, as shown in the attached instructions. Figure 3As shown in Figure 3d. Without discarding the original culture medium, half the volume of N2B27 electroporation reprogramming medium (pre-added with 0.25 mM sodium butyrate, 5 μM PS48, and 2 μM IHMT-MST1-39) was added to the culture plate. The cells were then transferred to a 37°C, 5% CO2 incubator for N2B27 adaptation culture. During this period, half of the N2B27 electroporation reprogramming medium was replaced every 24 hours to maintain the stability of the culture environment and cell viability. It should be noted that some cell loss may occur during medium replacement. To reduce cell loss, the collected medium can be centrifuged, and the centrifuged cells can be added back to the culture plate.

[0040] 4.3 Complete conversion and hypoxia induction: Following adaptation culture in N2B27 electroporation reprogramming medium (day 3 after electroporation), the culture environment needs to be adjusted to accelerate the reprogramming process. First, half of the old medium is gently aspirated, and an equal volume of N2B27 complete reprogramming medium containing 0.25 mM sodium butyrate and 5 μM PS48 is added. Then, the culture plates are transferred to a hypoxic incubator (1–3% O2, 5% CO2, 37°C) and cultured for 48 hours. The hypoxic environment significantly promotes the initiation and efficiency of early reprogramming events by modulating intracellular signaling pathways, regulating cell metabolism, and reducing oxidative stress. At this stage, cells are in a more favorable environment for reprogramming, accelerating the transition to a pluripotent state.

[0041] 4.4 iPSC clonal formation under normoxic conditions: After culturing in N2B27 electroporation reprogramming medium under hypoxic conditions for 48 hours, the culture environment needs to be converted to normoxic conditions to promote further development and stability of iPSC-like cells. On day 5 after cell electroporation, half of the N2B27 medium containing 5 μM PS48 was replaced to ensure the freshness of the medium components. Subsequently, the culture plates were transferred to a normoxic incubator at 37°C and 5% CO2 for continued culture. This stage of culture aims to promote the formation and stability of iPSC-like cells, providing high-quality cell resources for subsequent cell screening and application. Through this series of adaptive culture steps, this invention ensures the stable growth and efficient reprogramming of reprogrammed cells under different environments, providing a reliable technical guarantee for ultimately obtaining iPSCs with high pluripotency characteristics.

[0042] Example 5: The reprogramming scheme of the present invention for the late-stage cell expansion and iPSC cloning stage 5.1 Preliminary conversion of culture medium and induction of colony formation: In Example 4, cells were cultured using N2B27 electroporation reprogramming medium. At this point, the cells had initially adapted to the reprogramming environment and begun to transition to a pluripotent state, as shown in the attached diagram. Figure 3 As shown in 3f. To further optimize culture conditions and improve the proportion and purity of iPSCs, the culture medium needs to be adjusted. Without affecting cell adhesion and growth, 50% of the old N2B27 reprogramming medium was gently aspirated, and an equal volume (based on the remaining medium volume in the well) of preheated iPSC complete medium was added (supplemented with 10 μM Y27632, 0.5 μM PD0325901, 5 μM CHIR99021, 2 μM SB431542, and 5 μM Hetrombopag). The cells were then cultured at 37°C, 5% CO2, and normoxic conditions. This phased transition aims to allow the reprogrammed cells to smoothly adapt to the pluripotent stem cell maintenance environment and avoid cell detachment or death caused by drastic changes in culture medium composition.

[0043] 5.2 Gradual fluid replacement strategy (half-volume fluid replacement): Within 72 hours of replenishing half the volume of iPSC complete medium, a half-volume medium replacement strategy should be used for culturing. Every 24 hours, gently aspirate the upper half of the medium from the wells, avoiding disturbing cells that are not firmly attached at the bottom, and slowly add an equal volume of fresh, preheated iPSC complete medium. During this process, special care must be taken to handle the cells gently to avoid physical damage. At this stage, the cells are in a critical reprogramming phase and have poor adhesion stability; therefore, pipetting or centrifugation to recover the cells is strictly prohibited. When removing the medium, it is recommended to use a small-capacity pipette to gently aspirate the surface medium multiple times to minimize interference with the cells. When replenishing the medium, the movements should be gentle to prevent damage to cells that are about to attach.

[0044] 5.3 Increasing the fluid replacement ratio (replacing 2 / 3 of the fluid volume): After a 72-hour half-volume medium replacement, the medium replacement ratio was gradually increased to 2 / 3 volume over the next 4-5 days. Around Day 15, adherent iPS-like cells could be observed under a microscope, as shown in the attached image. Figure 4 As shown in 4b. Every 24 hours, gently aspirate 2 / 3 of the old culture medium from the well and replenish with an equal volume of fresh iPSC complete culture medium. Continue to emphasize gentle handling during this stage to avoid unnecessary physical interference with the cells. Gradually increasing the medium replacement ratio can further optimize the culture environment, effectively remove cellular metabolic waste, and provide sufficient nutrients to support clonal expansion.

[0045] 5.4 Complete fluid replacement and iPSC maintenance: After several half-volume and two-thirds volume medium replacements, the cells have gradually adapted to the new culture environment, at which point a complete medium replacement strategy can be adopted. Every 48 hours, all the culture medium in the wells is aspirated, and the cells are gently washed once with pre-warmed PBS, followed by the addition of sufficient fresh iPSC complete medium. Complete medium replacement thoroughly refreshes the culture system, providing an optimal growth environment for the established iPSC clones, promoting their further proliferation and maintaining their pluripotency. At this stage, the cells exhibit high pluripotency characteristics, as shown in the attached figure. Figure 4 As shown in 4d, obvious iPSC clonal clusters are visible, and a complete change of medium will further promote their transformation into stable iPSCs.

[0046] Example 6: Identification of iPSC pluripotency In Example 5, approximately 28 to 30 days after electroporation, iPSC clones with typical morphology, clear boundaries, large nuclei, and dense cytoplasm appeared in the culture plate (the typical morphology is shown in the attached figure). Figure 4 As shown in Figure 4f), single-clone picking can then be performed. The target clone is isolated using mechanical or collagenase methods and transferred to fresh 6-well plates coated with VTN. Amplification culture is then performed using the iPSC complete medium as described in Example 5. Once the clonal clusters have grown to a suitable size (as shown in Figure 4f), single-clone picking can be performed. Figure 4 4g of the sample was used for pluripotency identification. Immunofluorescence staining was used to detect the expression of core pluripotent transcription factors and surface markers. Figure 5 As shown, the iPSC clones obtained by the method of this invention highly express pluripotent proteins such as OCT4, SOX2, NANOG, and Klf4, and the expression is correctly localized. The cell clusters obtained using the culture system of this invention have typical iPSC morphological characteristics, highly express pluripotency-related markers, and have a stable background, confirming that they are induced pluripotent stem cells with complete pluripotency.

[0047] Comparative Example 1: Traditional PBMC isolation and culture protocol Comparative Example 1 uses a traditional PBMC culture protocol, and its main difference from Example 2 of the present invention is that the culture medium does not contain the specific combination of cytokines provided by the present invention.

[0048] 1.1 Performance of fresh PBMCs in basal medium The PBMC isolation method was the same as in Example 2, using Ficoll density gradient centrifugation to obtain cells from fresh peripheral blood. The isolated cells were seeded into two culture systems: (A) Basic culture system: Classic X-VIVO 15 medium supplemented with 100 ng / mL SCF and 100 ng / mL FLT3L, cultured at normoxic conditions (21% O2, 5% CO2, 37℃) for 96 hours, with half-volume medium replacement every 24 hours. (B) Invention culture system: Operated according to the method provided in Example 2, with specific cytokines provided by the invention added to the culture medium and hypoxia-noroxic timing regulation employed.

[0049] As attached Figure 2 The flow cytometry results at 2d and 2e showed that the proportion of CD34⁺ cells in the basal culture system was low, and after 96 hours of culture, the proportion was far below 20%. This indicates that without the support of key cytokines such as UM171, TPO, ANGPTL3, and TFF3, and under hypoxia-induced conditions, the target hematopoietic stem / progenitor cells could not survive and expand effectively, and most of the cells underwent differentiation or apoptosis.

[0050] 1.2 Comparison of amplification capacity of cryopreserved PBMCs in different culture systems To further verify the strong support effect of the system of the present invention on cell state, an additional set of experiments using cryopreserved and thawed PBMCs was added to this comparative example. Fresh PBMCs from the same source were cryopreserved in liquid nitrogen for 3 months using programmed cryopreservation solution and then thawed, and were inoculated into the above-mentioned (A) basic culture system and (B) the culture system of the present invention, respectively.

[0051] The experimental comparison results are attached. Figure 2 As shown in Figures 2a-c, cryopreservation significantly impaired the activity and function of PBMCs. In the basal culture system (A), the CD34⁺ cell expansion capacity of cryopreserved PBMCs was significantly reduced, with the post-culture ratio approaching that of fresh PBMCs in their initial state. However, in the culture system of this invention (B), although the initial state of cryopreserved PBMCs was far inferior to that of fresh PBMCs, their CD34⁺ cell expansion still achieved a much greater effect than that of the basal system. This result not only reaffirms the effectiveness of the cytokine combination and culture conditions of this invention but also highlights its application potential in handling non-ideal starting materials (such as cryopreserved samples) while still demonstrating significant advantages.

[0052] Comparative Example 2: Electroporation Reprogramming and Initial Culture of Traditional Culture Protocol PBMCs cultured in the basic culture system of Comparative Example 1 were collected, and their cell states are shown in the attached figure. Figure 3As shown in 3a, the cells exhibit poor morphology and abundant fragmentation. Subsequently, electroporation was performed using the exact same electroporation parameters as in the embodiments of the present invention (1650V, 10ms, 3 pulses) and an equal volume of Epi5 reprogrammed vector. After electroporation, the cells were seeded in VTN-coated culture plates at the same coating concentration as in the embodiments of the present invention. However, after seeding, the cells were cultured in classic medium (X-VIVO 15 medium supplemented with 100 ng / mL SCF and 100 ng / mL FLT3L) for 24 hours under normoxic conditions (21% O2, 5% CO2, 37°C).

[0053] Due to the lack of support from key small molecule components such as α-tocopherol added in this invention, the electroporated cells exhibited severe stress responses. Compared to the cells cultured in Example 4 of this invention (see Appendix...),... Figure 3 Compared to the "clear cell bodies and intact morphology" of cells cultured in Comparative Example 2 (see Appendix 3), which exhibited an active metabolic state, the cells cultured in Comparative Example 2 (see Appendix 3) showed a significantly different appearance. Figure 3 In cell 3c), the cells exhibited shrunken morphology, poor refractive properties, and visible cell debris in the background, indicating a significantly increased apoptosis rate. This result directly demonstrates that providing only a physical coating matrix without the necessary biochemical signal support is insufficient to maintain cell survival and homeostasis after electroporation, thus severely hindering the initiation of the reprogramming process.

[0054] Comparative Example 3: Reprogramming Induction and Maintenance of Traditional Culture Protocols In Comparative Example 2, PBMCs were electroporated for 24 hours (i.e., the day after electroporation). Half of the culture medium was replaced with basic N2B27 medium without sodium butyrate, PS48, and IHMT-MST1-39, and cultured entirely under normoxic conditions (21% O2). Due to the lack of synergistic regulation of these key small molecules on critical reprogramming signaling pathways, the initiation phase of reprogramming was severely inhibited. (See attached...) Figure 3 As shown in 3e, the cells failed to adhere effectively or form early clonal structures at this stage, and no obvious morphological changes were observed, indicating that the reprogramming process was blocked.

[0055] Approximately 10 days after electroporation, the culture medium was replaced with a simplified StemFlex medium containing only 10 μM Y27632, which does not contain the key small molecules needed to maintain the pluripotent network. Throughout the subsequent culture, to maintain an extremely limited number of adherent cells, a conservative strategy of half-volume medium replacement every 24 hours was adopted, and a complete medium replacement was never performed. This insufficient culture support led to the continuous accumulation of metabolic waste, gradual depletion of nutrients, and a continuous deterioration of the culture environment. (See attached...) Figure 4As shown in 4a, 4c, and 4e, compared to the results of the embodiments of the present invention, the cells in this comparative example failed to form typical, well-defined iPSC clones within 4 weeks. Even when a very small number of cell clusters appeared, their growth was extremely slow, making it difficult to effectively expand and mature, ultimately failing to obtain high-quality pluripotent stem cells.

[0056] Compared to the culture method of this invention, traditional culture methods yield very few iPSC clones with a reprogramming efficiency of less than 0.1%. The culture method of this invention significantly increases the reprogramming efficiency by more than 20%, and in the examples, the iPSC induction efficiency can be improved by more than 40%. Figure 6 As shown, in traditional culture protocols, the emergence of typical clones is delayed until more than 20 days after electroporation, and they exhibit heterogeneous morphology and blurred boundaries. This result fully demonstrates the necessity and outstanding advantages of the hypoxic environment, specific cytokine combination, and time-sequential intervention of small molecule additives used in this invention for the efficient and stable generation of high-quality iPSCs.

[0057] In summary, this invention provides a highly efficient, stable, and reproducible systematic solution for reprogramming PBMCs into induced iPSCs. Through the synergistic effect of three core innovations, it successfully solves the problems of low reprogramming efficiency, unstable cell quality, and high operating costs inherent in traditional methods: 1. Innovative CD34⁺ cell pre-expansion system: Through the combined application of a specific combination of cytokines (UM171, SCF, ANGPTL3, TPO, Flt3L, IL-3, IL-6, TFF3) and dynamic oxygen concentration regulation (hypoxia → normoxic), the proportion of CD34⁺ cells can be increased to ≥20% within 4 days, providing a high-quality and sufficient source of starting cells for reprogramming.

[0058] 2. Optimization of the electroporation microenvironment and reprogramming initiation: The strategy of coating cells with VTN matrix and adding α-tocopherol immediately after electroporation significantly alleviated cell stress caused by electroporation. Furthermore, by sequentially adding sodium butyrate, PS48, and IHMT-MST1-39 to N2B27 medium and inducing hypoxia during the critical window period (days 3-5), the reprogramming-related signaling pathways were effectively activated, creating the necessary conditions for pluripotency conversion.

[0059] 3. Efficient support system for iPSC clone formation and stability: By progressively switching from N2B27 medium to a well-defined iPSC complete medium (containing Y27632, PD0325901, CHIR99021, SB431542, and Hetrombopag), a stable pluripotency network is established. Combined with a refined culture strategy of switching from half-volume to complete medium replacement, an optimal environment is provided for the formation, amplification, and long-term stable maintenance of iPSC clones.

[0060] Based on the combined data from the embodiments and comparative examples, this invention ultimately achieves a significant improvement in reprogramming efficiency compared to traditional methods, with improvements exceeding 40% in the embodiment data. Furthermore, the obtained iPSC clones exhibit high positive expression of pluripotency markers. The entire process utilizes a serum-free, well-defined culture system, with batch-to-batch variability of less than 5%, completely avoiding the risks and uncertainties associated with exogenous serum and demonstrating extremely high reproducibility.

[0061] In summary, this invention establishes for the first time a complete standardized process from PBMC source control, pretreatment amplification, reprogramming induction to iPSC establishment, providing an efficient, safe, and stable cell preparation platform for basic research on reprogramming technology, disease model construction, drug screening, and future personalized cell therapy. It has significant application value and industrialization prospects in the field of regenerative medicine.

[0062] This invention describes specific implementation schemes in detail through examples and comparative examples. The above examples are only used to illustrate the technical solutions of this invention, and are not intended to limit it. Those skilled in the art can adjust and optimize the implementation schemes under the guidance of this invention without departing from the core idea of ​​the invention. All technical solutions obtained by replacing the same or equivalent technical means are within the scope of protection of the claims of this invention.

Claims

1. A method for improving the efficiency of reprogramming a PBMC to an iPSC, characterized in that, The following cultivation steps are included in both hypoxic environments (1–3% O2, 5% CO2) and normoxic environments (20–22% O2, 5% CO2): S1. PBMC pretreatment and CD34⁺ cell expansion culture: PBMCs were isolated from freshly collected peripheral blood and seeded into non-tissue culture dishes. CD34⁺ cells were expanded using serum-free PBMC expansion medium supplemented with multiple cytokines UM171, SCF, ANGPTL3, TPO, Flt3L, IL-3, IL-6 and TFF3. After 4 days of culture, the proportion of CD34⁺ cells was ≥20%. The first 48 hours were in a hypoxic environment and the next 48 hours were in a normoxic environment. 50% of the culture medium was replaced every 24 hours. S2. Efficient PBMC Reprogramming and iPSC Cloning Induction: PBMCs pre-expanded with CD34⁺ cells cultured in step S1 were electroporated to deliver reprogramming factors. The electroporated PBMCs were seeded in glassine-coated culture dishes, and reprogramming induction was achieved through staged medium changes: PBMC electroporated cells were successively adapted using PBMC expansion medium supplemented with 0.01–0.05 mM α-tocopherol; PBMC electroporated cells were induced to undergo N2B27 induction culture using N2B27 electroporation reprogramming medium, with sodium butyrate, PS48, and IHMT-MST1-39 added to the medium for reprogramming induction; PBMC electroporated cells were induced to develop iPSCs using complete iPSC medium containing Y27632, PD0325901, CHIR99021, SB431542, and Hetrombopag, ultimately obtaining iPSC clones.

2. The method according to claim 1, characterized in that, The serum-free PBMC amplification medium used in step S1 is X-VIVO 15 basal medium. The cytokines added to the medium and the amounts added are as follows: 15-25 ng / mL UM171, 45-55 ng / mL SCF, 95-105 ng / mL ANGPTL3, 5-15 ng / mL TPO, 45-55 ng / mL Flt3L, 15-25 ng / mL IL-3, 15-25 ng / mL IL-6, and 50-100 ng / mL TFF3.

3. The method according to claim 1, characterized in that, In step S2, the process of reprogramming induced PBMCs after electroporation through phased culture medium conversion is as follows: (1) Adaptive culture of PBMC electroporation cells: On the day of electroporation, PBMC amplification medium supplemented with 0.01~0.05mM α-tocopherol was used for 24 hours of adaptive culture under normoxic conditions; (2) N2B27-induced culture of PBMC electroporated cells: On the day after electroporation, half of the culture medium was replaced with N2B27 electroporation reprogramming medium supplemented with 0.2-0.3mM sodium butyrate, 5-10 μM PS48 and 1-3 μM IHMT-MST1-39, and the cells were cultured under normoxic conditions; On the 3rd and 4th days of electroporation, half of the culture medium was replaced with N2B27 electroporation reprogramming medium supplemented with 0.2-0.3mM sodium butyrate and 5-10 μM PS48 every 24 hours; Starting from the 5th day of electroporation, half of the culture medium was replaced with N2B27 electroporation reprogramming medium supplemented with 5-10 μM PS48 every 24 hours; From the 3rd to the 5th day of electroporation, hypoxia-induced culture was carried out in a hypoxic environment for 2-3 days, and thereafter the cells were cultured under normoxic conditions. (3) PBMC electroporation to induce iPSC culture: On days 9-10 of electroporation, replace 50% of the N2B27 reprogramming medium with iPSC complete medium. Within 72 hours after the medium change, replace half of the iPSC complete medium every 24 hours. In the next 24-48 hours, replace 2 / 3 of the iPSC complete medium every 24 hours. After that, replace the entire iPSC complete medium every 48 hours.

4. The method according to any one of claims 1 and 3, characterized in that, The complete iPSC culture medium contains 9–14 μM Y27632, 0.5–1 μM PD0325901, 5–10 μM CHIR99021, 2–5 μM SB431542, and 5–10 μM Hetrombopag; and after iPS cells appear during the culture process, the concentration of Y27632 in the replaced complete iPSC culture medium is adjusted to 10 μM.

5. The method according to claim 4, characterized in that, The iPSC complete medium was prepared by adding 9–14 μM Y27632, 0.5–1 μM PD0325901, 5–10 μM CHIR99021, 2–5 μM SB431542 and 5–10 μM Hetrombopag to StemFlex complete medium.

6. The method according to claim 1, characterized in that, In step S2, the electrically transferred PBMC operates at a speed of 1 to 2 × 10⁻⁶. 5 Cells / wells were seeded at a density of 1.5–2 μg / cm² in 6-well plates coated with phyllin. 2 .

7. The method according to claim 1, characterized in that, The pre-expansion seeding amount of the PBMCs was 5 × 10⁻⁶ cells. 4 ~5×10 5 cells / mL.

8. The method according to claim 1, characterized in that, The PBMCs obtained after peripheral blood separation in step S1 are 5×10 4 ~5×10 5 The cells / mL were seeded onto the culture surface of non-tissue culture vessels.

9. The method according to claim 1, characterized in that, In step S2, the reprogramming factor is delivered to the PBMC via electrotransfer. This is done using a Neon electrotransfer instrument in a buffer containing 10% Ficoll PM400. The non-integrative Epi5 attach vector preloaded with the OCT4, SOX2, KLF4, c-MYC, mp53DD, and EBNA1 genes is electrotransferred into the PBMC to complete the plasmid transfection for reprogramming.

10. The method according to claim 1, characterized in that, In step S1, fresh peripheral blood is separated into PBMCs by Ficoll density gradient centrifugation. The time from peripheral blood collection to the completion of PBMC separation does not exceed 4 hours, and the blood is kept at 2-8°C throughout the process to maintain cell biological activity.

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