Micromagnetic resonance relaxometry (MRR) for rapid and non-invasive detection of iPSC quality and differentiation

Abstract

A method and system for assessing the quality and differentiation of induced pluripotent stem cells using T2 magnetic resonance relaxometry.

Description

[Technical Field]

[0001] Priority claims This application claims priority to U.S. Provisional Patent Application No. 63 / 4q12,253, filed October 3, 2022, which is incorporated by reference in its entirety.

[0002] The present invention features systems and methods for stem cell analysis. [Background technology]

[0003] Magnetic resonance relaxometry (MRR) devices are capable of measuring the T2 relaxation time of microliter samples. For example, Peng,WK,et al.,2020.Molecular phenotyping of oxidative stress in diabetes mellitus with point-of-care NMR system.NPJ Aging and Mechanisms of Disease,2020,6(1),pp.1-12;Fook Kong,T.,et al.Enhancing malaria diagnosis through microfluidic cell enrichment and magnetic resonance relaxometry detection.Scientific Reports,2015,5(1),pp.1-12;Peng,WK,et al.Development of miniaturized,portable magnetic resonance relaxometry system for point-of-care medical diagnosis.Review of Scientific Instruments,2012 83(9),p.095115;and Thamarath,SS,et al.2022.Rapid and Live-cell Detection of Senescence in Mesenchymal Stem Cells by Micro Magnetic Resonance Relaxometry.bioRxiv doi.org / 10.1101 / 2022.06.01.494362, each of which is incorporated by reference in its entirety. Briefly, MRR devices measure the relaxivity from spin echoes of cellular water content using the same principles as magnetic resonance imaging (MRI) or nuclear magnetic resonance (NMR), with the distinct advantage of being able to use microliter-sized samples and minimal cell number requirements (approximately 110 to approximately 180 k cells per test), making them suitable for cell therapy situations. [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Peng,WK,et al.,2020.Molecular phenotyping of oxidative stress in diabetes mellitus with point-of-care NMR system.NPJ Aging and Mechanisms of Disease,2020,6(1),pp.1-12 [Non-patent document 2] Fook Kong,T.,et al.Enhancing malaria diagnosis through microfluidic cell enrichment and magnetic resonance relaxometry detection.Scientific Reports,2015,5(1),pp.1-12;Peng,WK,et al.Development of miniaturized,portable magnetic resonance relaxometry system for point-of-care medical diagnosis.Review of Scientific Instruments,2012 83(9),p.095115 [Non-patent document 3] Thamarath,SS,et al.2022.Rapid and Live-cell Detection of Senescence in Mesenchymal Stem Cells by Micro Magnetic Resonance Relaxometry.bioRxiv doi.org / 10.1101 / 2022.06.01.494362 Summary of the Invention

[0005] In general, magnetic resonance relaxometry (MRR) measurements can be used to evaluate induced pluripotent stem cells (iPSCs) and cells differentiated from them. For example, measurement of MRR, especially T2-dependent characteristics, can be used to identify iPSCs with superior chondrogenic or cartilage regenerative capabilities. Cell types can be differentiated by their intracellular Fe 3+ The differentiation of SCPCs can be achieved through their content. For example, OCT4 levels in SCPCs can be measured using the MRR system and were investigated. This measurement allows for evaluation of the differentiation efficiency endpoint between batches of SCPCs. Furthermore, T2 measurement of iPSCs as early as day 1 in the differentiation process can be used to predict the differentiation efficiency of iPSCs to SCPCs.

[0006] In one embodiment, a method for evaluating induced pluripotent stem cells and cells differentiated therefrom may include loading a sample containing a plurality of induced pluripotent stem cells and cells differentiated therefrom into a sensor, placing the sensor containing the sample within or near a detection coil of a magnetic resonance relaxometry device, determining the T2 value of the sample, and evaluating the quality of the induced pluripotent stem cells and cells differentiated therefrom from the T2 value.

[0007] In another embodiment, the system for evaluating induced pluripotent stem cells and cells differentiated therefrom can include a magnetic resonance relaxometry device, which is configured to determine the T2 value of a sample containing a plurality of induced pluripotent stem cells and cells differentiated therefrom in a sensor, and to monitor the T2 value of the sample, thereby evaluating induced pluripotent stem cells.In certain circumstances, the system can include a cell passage device as a source of a plurality of induced pluripotent stem cells and cells differentiated therefrom.For example, the T2 value of passaged induced pluripotent stem cells and cells differentiated therefrom can be measured to evaluate the variation of passaged induced pluripotent stem cells and cells differentiated therefrom from induced pluripotent stem cells and cells differentiated therefrom.

[0008] In certain circumstances, the evaluating may include comparing the T2 values ​​of the induced pluripotent stem cells and cells differentiated therefrom with previously determined T2 values ​​of the induced pluripotent stem cells and cells differentiated therefrom.

[0009] In certain circumstances, T2 values ​​can correlate with the proportion of differentiated progenitor cells.

[0010] In certain circumstances, the T2 value can correlate with the proportion of remaining undifferentiated iPSCs in a differentiated cell population.

[0011] In certain circumstances, the T2 value can correlate with the ability of iPSCs to differentiate into progenitor or fully differentiated cells.

[0012] In certain circumstances, T2 values ​​can be correlated with EdU colony formation assays.

[0013] In certain situations, assessing the quality of the induced pluripotent stem cells and cells differentiated therefrom based on T2 values ​​is performed on day 1 of the differentiation process. In certain situations, assessing the quality of the induced pluripotent stem cells and cells differentiated therefrom based on T2 values ​​is performed on day 4 of the differentiation process. In certain situations, assessing the quality of the induced pluripotent stem cells and cells differentiated therefrom based on T2 values ​​is performed on day 10 of the differentiation process. In other situations, assessment can be performed on a combination of days 1 and 10, days 1 and 4, days 4 and 10, or days 1, 4, and 10.

[0014] In certain circumstances, induced pluripotent stem cells and cells differentiated therefrom are suitable for spinal cord injury cell therapy applications.

[0015] In certain circumstances, the method may include passaging the induced pluripotent stem cells and cells differentiated therefrom, and measuring the T2 values ​​of the passaged induced pluripotent stem cells and cells differentiated therefrom to assess variation of the passaged induced pluripotent stem cells and cells differentiated therefrom from the induced pluripotent stem cells and cells differentiated therefrom.

[0016] In certain circumstances, the sample may be a pellet containing induced pluripotent stem cells and cells differentiated therefrom in a sensor.

[0017] In certain circumstances, the sample may include clusters of induced pluripotent stem cells and cells differentiated therefrom.

[0018] In certain circumstances, the clusters may have a size of at least 50 microns.

[0019] In certain circumstances, T2 values ​​can correlate with the doubling time of induced pluripotent stem cells and cells differentiated therefrom.

[0020] In certain circumstances, T2 values ​​can correlate with differentiated stem cells.

[0021] In certain circumstances, T2 values ​​can be correlated with OCT4 expression.

[0022] In certain circumstances, the sensor may be a tube or a chamber.

[0023] In certain circumstances, the magnetic resonance relaxometry device may include a radio frequency probe.

[0024] In certain circumstances, determining the T2 value may involve delivering a pulse train for a period of less than one minute.

[0025] In certain circumstances, determining the T2 value can include acquiring and averaging between 2 and 70 scans, between 4 and 60 scans, or between 6 and 40 scans. For example, determining the T2 value can include acquiring and averaging between 10 and 30 scans.

[0026] In certain circumstances, the detection volume of a magnetic resonance relaxometry device can contain a sample volume of less than about 1 μL. For example, the volume can be less than 0.1 μL, less than about 0.01 μL, less than about 0.001 μL, or less than about 0.0001 μL, and in certain circumstances, the volume can be between about 1 pL and 10 pL.

[0027] In certain circumstances, the liquid sample may be free of paramagnetic or ferromagnetic materials, such as paramagnetic or ferromagnetic metal ions or compounds thereof.

[0028] In certain circumstances, the cell passaging device can be a source of a plurality of induced pluripotent stem cells and cells differentiated therefrom. For example, the cell production device can include a cell culture system.

[0029] In certain circumstances, the liquid sample can be contained in a microcapillary.

[0030] Other aspects, embodiments, and features will become apparent from the following description, drawings, and claims. [Brief explanation of the drawings]

[0031] [Figure 1] 1 depicts a schematic diagram of a magnetic resonance relaxometry (MRR) instrument. [Figure 2] 1 depicts a schematic diagram of a system including a magnetic resonance relaxometry (MRR) device and a cell production device as a source of stem cells. [Figure 3] Figures 3A-3B show the Carr-Purcell-Meiboom-Gill (CPMG) pulse sequence for measuring T2 values, and Figure 3C is a schematic diagram depicting the spin-spin relaxation time of iPSCs changing as they differentiate into SCPCs. [Figure 4] FIG. 1 is a schematic depicting the sample preparation process for iPSCs and spinal cord progenitor cells (SCPCs) being tested in the MRR instrument in suspension form. [Figure 5]FIG. 1 is a schematic depicting the sample preparation process for testing iPSCs and SCPCs on the MRR instrument in the form of pellets. [Figure 6]

[0023] Figure 1 is a graph depicting T2 relaxation times over several passages under normal culture conditions. Data were accumulated over 6 months of iPSCs generated herein. [Figure 7]

[0033] Figure 1 is a graph depicting T2 relaxation times over several passages under normal, controlled single-cell culture conditions. One culture was split into two separate cultures. Each culture was grown across eight different wells and seeded at a density of 500k cells per well. After three days of growth, a portion of the cells were harvested for magnetic testing using Method 1 and the cells were counted, and the remainder were reseeded for the next passage. [Figure 8] 1 is a graph depicting T2 relaxation times over several passages under normal controlled culture conditions. [Figure 9] 1 is a graph depicting the T2 relaxation times measured by method 2 for three cultures plotted against population doubling time. [Figure 10] Figure 1 depicts the T2 relaxation time of continuously passaged iPSCs versus frozen and thawed iPSCs. In this experiment, the magnetic measurements on the left were for cells continuously passaged from P38 to P40. The measurements on the right were obtained from cells that underwent cryopreservation. This process involves storing cells in medium, ROCKi, and DMSO at -80°C to acclimate them before transferring them to liquid nitrogen for long-term storage. The cells are then thawed, replated, and expanded according to the usual protocol. [Figure 11] 1 is a graph depicting the correlation between the percentage of large clumps seeded in culture and population doubling time. [Figure 12] 1 is a graph depicting OCT4 vs. MRR T2 relaxation time values ​​at day 10. As OCT4 values ​​decrease, T2 relaxation time values ​​increase. [Figure 13]1 is a graph depicting residual OCT4 expression at day 10 versus T2 relaxation time at day 1. Prediction of OCT4 values ​​at day 10 based on MRR measurements at day 1. [Figure 14] Figures 14A-14E depict the changes in expression of OCT4 and SOX1, as well as T2 measurements, as iPSCs differentiated into SCPCs. Figure 14A shows immunofluorescence staining to illustrate the changes in Oct4 and Sox1 phenotype during the differentiation of iPSCs into SCPCs over three different time points (days 1, 4, and 10). DAPI (blue, nuclei), SOX1 (green, neural progenitor marker), and OCT4 (red, pluripotency marker) (Scale bar: 50 μm). Figure 14B shows a decrease in OCT4+ cells as iPSCs differentiate into SCPCs at day 10. Figure 14C shows an increase in T2 as iPSCs differentiate into SCPCs at day 10. Figure 14D shows an increase in T2 along with a decrease in the level of OCT4+ cells over the 10-day differentiation period. Figure 14E shows iPSCs and SCPCs stained with the Fe3+ reversible fluorescent sensor CLEC23, and FACS analysis shows that the fluorescence intensity of stained cells corresponds to their T2 (n = 2). For each replicate experiment, at least 10 ROIs (more than 60,000 cells) were quantified (n = 4). Statistical significance was determined by the nonparametric Kruskal-Wallis test followed by Dunn's post-hoc test. [Figure 15]Figures 15A-15C illustrate that the presence of artificially spiked iPSCs in SCPC populations reduces the measured T2 values ​​in a dose-dependent manner. Figure 15A shows artificial spiking of iPSCs in SCPCs at concentrations of 1%, 5%, and 10% (n=4). Figure 15B shows immunofluorescence staining of SCPCs subjected to a colony culture assay with spiked iPSCs. Staining includes DAPI (blue, nuclei) and OCT4 (red, pluripotency marker). Increases in the number and size of iPSC colonies were observed with increasing spiked iPSC concentrations (n=2). Figure 15C shows quantification of iPSC colony size as a frequency distribution plot of colony area size (n=2). For each replicate experiment, at least 100 ROIs (over 100,000 cells) were quantified (n=3). Statistical significance was determined by the Mann-Whitney u test. [Figure 16] Figures 16A-16C depict the correlation between T2 at day 10 and OCT4+ levels, the correlation between T2 at day 1 and day 10, and the correlation between T2 at day 1 and day 10 and OCT4+ levels at day 10. Pearson's correlation coefficient, a statistical measure of the strength of the linear relationship between paired data, was used to benchmark T2 relaxation time measurements for OCT4+ cells quantified by immunofluorescence staining. Figure 16A shows the correlation between SCPC T2 at day 10 and the percentage of OCT4+ cells quantified by immunofluorescence staining at day 10. Figure 16B shows the correlation between undifferentiated iPSC T2 at day 1 and SCPC T2 at day 10. Figure 16C shows the correlation between undifferentiated iPSC T2 at day 1 and the percentage of OCT4+ cells quantified by immunofluorescence staining at day 10. For each replicate experiment, at least 10 ROIs (over 60,000 cells) were quantified (n = 4). [Figure 17]Figures 17A-17G illustrate that a decrease in CHIR concentration results in poor differentiation efficiency of iPSCs to SCPCs, with a corresponding decrease in T2. ​​Figures 17A-17C show FACS analysis of SOX1+, Nestin+, and OCT4+ cells (n=3) with decreasing CHIR concentration. Figure 17A shows a decrease in SOX1+ cells with decreasing CHIR concentration. Figure 17B shows a decrease in Nestin+ cells with decreasing CHIR concentration. Figure 17C shows an increase in OCT4+ cells with decreasing CHIR concentration. Figure 17D shows a decrease in T2 with decreasing CHIR concentration (n=3). Figure 17E shows a decrease in T2 with decreasing SCPC SOX1+ levels (n=3). Figure 17F shows a decrease in T2 with decreasing SCPC Nestin+ levels (n=3). Figure 17G shows a decrease in T2 with increasing SCPC OCT4+ levels (n=3). Statistical significance was determined by independent paired two-tailed t-test. [Figure 18] Figures 18A-18C depict a comparison between CLEC23 iPSCs and BJ-iPSCs. Figure 18A shows that CLEC23 iPSCs and BJ iPSCs exhibit similar ranges of T2 relaxation times and OCT4 levels. Figure 18B shows that day 10 BJ-SCPCs have a higher percentage of SOX1+ cells and T2 relaxation times compared to day 10 CLEC23 SCPCs (n=3). Figure 18C shows that day 10 BJ-SCPCs have a lower percentage of OCT4+ cells and higher T2 relaxation times compared to day 10 CLEC23 SCPCs (n=3). For each replicate experiment, at least 25 ROIs (over 60,000 cells) were quantified (n=3). DETAILED DESCRIPTION OF THE INVENTION

[0032] As described herein and taking into account the above device description, a method for evaluating induced pluripotent stem cells and cells differentiated therefrom can include loading a sample containing a plurality of induced pluripotent stem cells and cells differentiated therefrom into a sensor, placing the sample-containing sensor inside or near the detection coil of a magnetic resonance relaxometry device, determining the T2 value of the sample, and evaluating the quality of the induced pluripotent stem cells and cells differentiated therefrom from the T2 value. The quality of iPSCs can be a measure of important quality characteristics of iSPCs. For example, population doubling time, cellular variability from passage to passage, and the degree of differentiation observed in iPSCs can each be important qualities in evaluating the health, nature, or status of iSPCs. A decrease in T2 can indicate weak or defective iSPCs.

[0033] For example, the emergence of induced pluripotent stem cells (iPSCs) has brought a promising solution for replacing damaged neurons and glial cells, especially in cases of spinal cord injury (SCI). Despite its advantages, iPSCs and their differentiation into neural progenitor cells is a variable process, driving the need to reliably assess the degree of differentiation achieved in any given batch of cells and to verify the quality and safety of the differentiated cells. In this study, human iPSCs were differentiated into spinal cord progenitor cells (SCPCs). During the 10-day differentiation process, the intracellular iron content (i.e., Fe 3+) was measured at different time points via its T2 relaxation time in a rapid and label-free manner using magnetic resonance relaxometry (MR). SCPC batches containing higher levels of pluripotency markers (i.e., OCT4) were found to have lower T2 (p<0.005) than SCPC batches with lower levels of these markers. Furthermore, by adversely affecting iPSC differentiation into SCPCs, groups with lower levels of neural progenitor (i.e., SOX1) and stem cells (i.e., Nestin) have lower T2 (p<0.005) than normally differentiated SCPCs. Our technology provides a rapid, label-free method for determining key quality characteristics in iPSC-derived progeny, ideally suited as a quality control tool in cell therapy manufacturing.

[0034] In another embodiment, the system for evaluating induced pluripotent stem cells and cells differentiated therefrom can include a magnetic resonance relaxometry device, which is configured to determine the T2 value of a sample containing a plurality of induced pluripotent stem cells and cells differentiated therefrom using a sensor, and to evaluate the induced pluripotent stem cells and cells differentiated therefrom by monitoring the T2 value of the sample.In certain circumstances, the system can include a cell passage device as a source of a plurality of induced pluripotent stem cells and cells differentiated therefrom.For example, the T2 value of passaged induced pluripotent stem cells can be measured to evaluate the variation of passaged induced pluripotent stem cells and cells differentiated therefrom from induced pluripotent stem cells and cells differentiated therefrom.

[0035] In certain circumstances, the evaluating may include comparing the T2 values ​​of the induced pluripotent stem cells and cells differentiated therefrom with previously determined T2 values ​​of the induced pluripotent stem cells and cells differentiated therefrom.

[0036] In certain circumstances, the method may include passaging the induced pluripotent stem cells and cells differentiated therefrom, and measuring the T2 values ​​of the passaged induced pluripotent stem cells and cells differentiated therefrom to assess variation of the passaged induced pluripotent stem cells and cells differentiated therefrom from the induced pluripotent stem cells and cells differentiated therefrom.

[0037] In certain circumstances, the sample can be a pellet containing induced pluripotent stem cells and cells differentiated therefrom, which can be placed on the sensor in a position to improve the detection signal for T2 measurement.

[0038] In certain circumstances, the sample can contain clusters of induced pluripotent stem cells and cells differentiated therefrom. For example, the sample can contain one or more clusters. The clusters can have a size of at least 50 microns, at least 55 microns, at least 60 microns, at least 65 microns, at least 70 microns, at least 75 microns, at least 80 microns, at least 85 microns, or at least 90 microns. In certain circumstances, the clusters can have a maximum size of 250 microns.

[0039] In certain circumstances, T2 value can be correlated with the doubling time of induced pluripotent stem cells and the cells differentiated therefrom.Doubling time can be an indicator of cell health.For example, the shortening of doubling time during the measured period can indicate that iSPCs may be differentiated and may no longer be suitable for specific purposes.

[0040] The systems and methods described herein relate to induced pluripotent stem cells and cells differentiated therefrom. For example, the induced pluripotent stem cells can be differentiated stem cells.

[0041] In certain circumstances, T2 values ​​can correlate with the proportion of differentiated progenitor cells.

[0042] In certain circumstances, the T2 value can correlate with the proportion of remaining undifferentiated iPSCs in a differentiated cell population.

[0043] In certain circumstances, the T2 value can correlate with the ability of iPSCs to differentiate into progenitor or fully differentiated cells.

[0044] In certain circumstances, T2 values ​​can be correlated with EdU colony formation assays.

[0045] In certain circumstances, the T2 value can be correlated with differentiated stem cells. For example, the T2 value or progression of the T2 value of a sample can lead to establishing a quality control threshold that can improve the efficiency of using the cells.

[0046] In certain situations, assessing the quality of the induced pluripotent stem cells and cells differentiated therefrom based on T2 values ​​is performed on day 1 of the differentiation process. In certain situations, assessing the quality of the induced pluripotent stem cells and cells differentiated therefrom based on T2 values ​​is performed on day 4 of the differentiation process. In certain situations, assessing the quality of the induced pluripotent stem cells and cells differentiated therefrom based on T2 values ​​is performed on day 10 of the differentiation process. In other situations, assessment can be performed on a combination of days 1 and 10, days 1 and 4, days 4 and 10, or days 1, 4, and 10.

[0047] In certain circumstances, induced pluripotent stem cells and cells differentiated therefrom are suitable for spinal cord injury cell therapy applications.

[0048] In certain circumstances, T2 values ​​can be correlated with protein expression from iSPCs or SCPCs. For example, expression of OCT4 can indicate healthy cell behavior.

[0049] An apparatus for performing magnetic resonance relaxometry is described, for example, in U.S. Patent No. 10,429,467, the entire contents of which are incorporated by reference. Referring to FIG. 1, the apparatus can include an MRR system. FIG. 1 is a schematic diagram of a magnetic resonance relaxometry (MRR) system 100 according to one embodiment of the present disclosure. The system 100 can include a field-programmable gate array-based (FPGA-based) radio frequency (rf) spectrometer for controlling the MRR system 100, a first direct digital synthesis module for generating radio frequency pulses, a transmitter (TRANS) for transmitting the generated radio frequency pulses to a radio frequency (rf) probe and detection coil 110, a receiver (RCVR) for receiving resonance information from the radio frequency probe, a first power amplifier (PA), a preamplifier (p-amp), a duplexer (Dup) for transmitting high-power excitation pulses to the rf probe in a transmit mode and separating the high-power excitation pulses from the receiver during a receive mode, and a magnet system 120. The sample 130 can be placed in a tube or chamber, such as a microcapillary tube, which can be placed in an RF detection coil, a sensor, or a search. In many embodiments, the FPGA-based RF spectrometer can include a pulse programmer (PPG) and a second direct digital synthesis (DDS) adapted to control the FPGA-based RF spectrometer. The second DDS can generate a fixed intermediate frequency (IF). The first DDS can be configured to generate a variable desired frequency.According to one aspect of the present disclosure, the FPGA-based spectrometer can use the design described in Takeda K. (2007), "A highly integrated FPGA-based nuclear magnetic resonance spectrometer," Rev Sci Instrum 78(3):033103; and / or Takeda K. (2008) "OPENCORE NMR: open-source core modules for implementing an integrated FPGA-based NMR spectrometer," Journal of Magnetic Resonance 192(2):218-229. The teachings of these two references are incorporated by reference in their entirety.

[0050] To facilitate the processing of information to and from the MRR system 100, the FPGA-based rf spectrometer can be coupled to at least one external electronic device, which can include, for example, a personal computer, a mobile phone, and / or a portable electronic tablet. The coupling between the MRR system 100 and the at least one external electronic device may be by at least one of USB, HDMI, and / or wireless connection means such as Wi-Fi and / or Bluetooth.

[0051] In a conventional NMR system, the primary cost of the instrumentation is the superconducting magnet (or permanent magnet) and the RF spectrometer. According to one embodiment of the present disclosure, the overall system cost can be less than $2,500. The FPGA chips ($1,000 each), external GHz clocks ($250 each), DDS (Analog Devices; AD9858, $400 each), 1-watt power amplifiers ($100), preamplifiers ($50), RCVRs (AD8343, $4 each), transformers (AD834, $20 each, AD8343), and USBs (FT2232D, $10 each) account for the majority of the cost. The costs of the major electronic components used are listed in parentheses. Other peripheral components, such as pin connectors (e.g., SMA), capacitors, RF switches, RF transformers, and RF filters, cost less than $10 each.

[0052] The MRR system 100 can be adapted to operate in various modes for detecting NMR-active nuclei, such as proton, fluorine, phosphorus, and carbon. The magnetic field used in each mode in which the MRR system 100 operates depends on which nuclei are to be detected. Depending on the mode of operation, the MRR system 100 can operate in a magnetic field of approximately 0.1 to 3 Tesla (T), which can correspond to frequencies of approximately 1 to 150 MHz. For example, when the MRR system 100 is operating in proton NMR mode, the magnetic field is approximately 0.76 T, which corresponds to a proton NMR frequency of approximately 31.9 MHz.

[0053] The MRR system 100 can be controlled by an FPGA-based rf spectrometer, including a pulse programmer and a second DDS. Compared to CMOS technology, FPGAs offer the advantage of being reprogrammable. FPGA-based rf spectrometers can be programmable using tools and software provided by vendors such as Altera Corporation, San Jose, California, USA, and Xilinx, Inc., San Jose, California, USA. In an exemplary embodiment, the FPGA chip can include an EP3C80F780C8N Cyclone III (Altera) embedded in a breadboard (ACM-202-80C8, HumanData, Japan). This chip has 81,000 logic elements and, when fully utilized, can generate three independent if outputs.

[0054] The pulse programmer can generate high-power excitation RF pulses, which then pass through a first power amplifier to generate optimized RF power for a duration of approximately 1 to 1000 microseconds to effectively excite all nuclei. The high-power RF pulses are transmitted to an RF probe, as further described herein.

[0055] In exemplary operation, the powers used for liquid-state and solid-state NMR are approximately 0.1 W to 10 W and approximately 100 W to 1000 W, respectively. While "powerful" power amplifiers are often essential in MRR systems, such amplifiers are often bulky and require high power consumption, thereby imposing serious limitations on field operation. For example, a novel and lightweight 1-watt power amplifier can be constructed on a 4 cm x 4 cm printed circuit board. A solenoidal microcoil (inner diameter 700 to 1000 μm, e.g., 750, 800, 850, 900, or 950 μm) can additionally be used to generate a strong oscillating magnetic field B1, which also picks up signals from free induction decay (FID) or spin echoes. By using a duplexer, the high-power excitation in the case of pulses transmitted to the RF probe in transmit mode can be separated from the receiver or detection coil 110 during receive mode. The FID / spin echo is then amplified by a preamplifier (AMP-75+, Mini Circuits, USA) with 20 dB gain and a noise figure of 2.83, and finally filtered by an appropriate low-pass filter before entering the receiver circuitry. The FID is the observable NMR signal generated by a non-equilibrium nuclear spin magnetization process centered around the static magnetic field (conventionally along the z-axis). This non-equilibrium magnetization can be induced by applying a resonant radio frequency pulse close to the Larmor frequency of the nuclear spins. The spin echo is a single 90-degree flip followed by a refocusing pulse after flipping with a 180-degree pulse on resonance.

[0056] The magnet system 120 may be portable and lightweight (e.g., approximately 60 g) and may be adaptable to generate a high static magnetic field. The magnet system 120 may include at least one magnet positioned adjacent to the RF probe. An alternative embodiment includes having at least two magnets positioned adjacent to the RF probe. The RF probe may be positioned between the at least two magnets. The magnet system 120 may include permanent magnets and / or electromagnets. Permanent magnets used in the magnet system 120 may include, for example, neodymium-based magnets.

[0057] The detection region of the magnetic resonance relaxometry device can contain a sample for detection with a volume of less than about 1 μl. For example, the sample can be provided in a sensor, such as a capillary or microcapillary tube or chamber. The sample can be sealed from the surrounding environment to reduce exposure to oxygen and other materials that may adversely affect the ability of the magnetic resonance relaxometry device to detect cells. The sample can include a buffer solution that does not contain any paramagnetic iron compounds, which may be important for obtaining accurate results.

[0058] Current μMRR configurations are only suitable for measuring cell suspensions in samples, for example. μMRR configurations can be designed to adhere cells in cell culture plates. The devices, systems, and methods described herein can be used to develop and improve cell therapies by simplifying the cell identification process and facilitating cell removal from cell populations through the use of this approach in combination with cell manipulation techniques.

[0059] Several approaches can be taken to improve the accuracy of T2 values. For example, determining T2 values ​​can involve acquiring and averaging multiple scans. Up to 100 (or more) scans can be averaged. The number of scans can be fewer than 80, 70, or 60. More typically, 10 to 50 scans can be averaged. As a minimum, under certain circumstances, 2, 4, 5, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, or 30 scans can be averaged.

[0060] The systems and methods described herein can be used to develop assays. The assays can be used as quality control assays during iPSC production. Alternatively, assays can be performed midway through cell production to monitor the progress of the culture and identify any deviations from the accepted range of values, allowing for early identification of defective batches. The assays can be used in conjunction with cell production devices, allowing for multiple iPSC sources. For example, a cell passaging device can include a cell culture system.

[0061] 2, the systems described herein can include a magnetic resonance relaxometry device 200 and a cell passaging device 240. For example, a system for identifying the chondrogenic potential of mesenchymal stem cells can include a magnetic resonance relaxometry device configured to determine the T2 value of a liquid sample and evaluate the induced pluripotent stem cells by monitoring the T2 value of the sample.

[0062] Referring to Figures 3A and 3B, the Carr-Purcell-Meiboom-Gill (CPMG) pulse sequence for measuring T2 values ​​can function efficiently in the inhomogeneous magnetic field generated by the permanent magnets of the MRR. A train of radiofrequency pulses is applied to the proton nuclei at a resonant frequency of 21.65 MHz with an inter-echo time interval of t echo, and is repeated for several thousand echoes until the nuclei relax over time. The height of this decaying echo over time is called the transverse relaxation time (T2).

[0063] Figure 1 shows a noninvasive and rapid magnetic resonance relaxometry (MRR) measurement for evaluating induced pluripotent stem cells. Figure 1 is a schematic diagram showing the MRR system, which consists of a permanent magnet that generates a strong magnetic field (B0). A built-in radio frequency (RF) detection probe is connected to an RF spectrometer. A sample of iPSCs in a microcapillary tube is placed in the RF detection coil for T2 measurement. Figure 1B shows a microcapillary tube containing a 4 μl cell sample within the 4 mm detection range of the RF detection coil. A typical cell number required is 60,000 cells inside the detection volume. The gray area below is a crysseal for sealing the microcapillary tube.

[0064] FIG. 2 depicts a schematic diagram of a system including a magnetic resonance relaxometry (MRR) device and a cell passaging device as a source of multiple induced pluripotent stem cells.

[0065] Figures 3A and 3B show a Carr-Purcell-Meiboom-Gill (CPMG) pulse sequence for measuring T2 values, which works efficiently in the inhomogeneous magnetic field generated by the permanent magnet of the μMRR. The radiofrequency pulse train consists of a series of pulses with an inter-echo time interval t エコー A pulse is applied to the proton nuclei at a resonant frequency of 21.65 MHz at 1000 kJ / s. This is repeated for thousands of echoes until equilibrium is reached. The decaying peak height of this successive echo over time is called the transverse relaxation time, T2. Figure 3C shows a schematic depicting the change in spin-spin relaxation time of iPSCs as they differentiate into SCPCs.

[0066] The development of induced pluripotent stem cells (iPSCs) by Yamanaka and colleagues has ushered in a revolutionary era of stem cell therapy. See K. Takahashi and S. Yamanaka, "Induction of Pluripotent Stem Cells from Mouse Embryonic and Adult Fibroblast Cultures by Defined Factors," Cell, vol. 126, no. 4, pp. 663-676, August 2006, incorporated by reference in its entirety. iPSCs have the same capabilities as embryonic stem cells (ESCs) and can proliferate indefinitely under appropriate conditions and differentiate into any somatic cell lineage. See S. Yamanaka, "Pluripotent Stem Cell-Based Cell Therapy—Promise and Challenges," Cell Stem Cell, vol. 27, no. 4, pp. 523-531, October 2020, incorporated by reference in its entirety. Furthermore, iPSCs serve as a desirable alternative to ESCs because ESCs are derived from embryos, their use can be controversial in various settings and are limited in supply. Therapeutic applications of iPSC-enabled cell and tissue regeneration currently undergoing clinical trials include Parkinson's disease, macular degeneration, heart failure, spinal cord injury, platelet transfusion, cartilage defects, and cancer immunotherapy. For example, see S. Yamanaka, "Pluripotent Stem Cell-Based Cell Therapy - Promise and Challenges," Cell Stem Cell, vol. 27, no. 4, pp. 523-531, October 2020; J. Takahashi, "iPS cell-based therapy for Parkinson's disease: A Kyoto trial," Regen. Ther., vol. 13, pp. 18-22, March 2020; IW Karas, L.R. Collins, and AACreasey,’A Stem Cell Journey in Ophthalmology:From the Bench to the Clinic’,Stem Cells Transl.Med.,vol.10,no.12,pp.1581-1587,Dec.2021、S.Miyagawa et al.,’Case report:Transplantation of human induced pluripotent stem cell-derived cardiomyocyte patches for ischemic cardiomyopathy’,Front.Cardiovasc.Med.,vol.9,p.950829,Aug.2022、K.Sugai et al.,’First-in-human clinical trial of transplantation of iPSC-derived NS / PCs in subacute complete spinal cord injury:Study protocol’,Regen.Ther.,vol.18,pp.321-333,Dec.2021、N.Sugimoto et al.,’iPLAT1:the first-in-human clinical trial of iPSC-derived platelets as a phase 1 autologous transfusion study’,Blood,vol.140,no.22,pp.2398-2402,Dec.2022、K.Abe et al.,’Engraftment of allogeneic iPS cell-derived cartilage organoid in a primate model of articular cartilage defect’,Nat.Commun.,vol.14,no.1,Art.no.1,Feb.2023、およびD.Hong et al.See, for example, "Preliminary results of an ongoing phase I trial of FT500, a first-in-class, off-the-shelf, induced pluripotent stem cell (iPSC)-derived natural killer (NK) cell therapy in advanced solid tumors," J. Immunother. Cancer, vol. 8, no. Suppl 3, Nov. 2020. iPSCs are particularly important for spinal cord injury (SCI), where current treatments severely lack recovery and primarily focus on surgery for spinal realignment and subsequent rehabilitation. See, for example, N. Nagoshi, O. Tsuji, M. Nakamura, and H. Okano, "Cell therapy for spinal cord injury using induced pluripotent stem cells," Regen. Ther., vol. 11, pp. 75-80, Jun. 2019, which is incorporated by reference in its entirety. A promising alternative for SCI treatment is the use of iPSC cell therapy to generate the cells necessary to replace damaged neurons and glial cells, ensuring effective spinal cord regeneration. See, for example, M. Nakamura and H. Okano, 'Cell transplantation therapies for spinal cord injury focusing on induced pluripotent stem cells', Cell Res., vol. 23, no. 1, Art. no. 1, Jan. 2013, which is incorporated by reference in its entirety. Several research groups have successfully demonstrated that transplanting iPSC-derived neural progenitor cells into rodent and monkey animal models of SCI results in the recovery of their motor function. See, for example, M. Nakamura and H. Okano, 'Cell transplantation therapies for spinal cord injury focusing on induced pluripotent stem cells', Cell Res., vol. 23, no. 1, Art. no. 1, Jan. 2013, which is incorporated by reference in its entirety.See Okano, ’Cell transplantation therapies for spinal cord injury focusing on induced pluripotent stem cells’, Cell Res., vol. 23, no. 1, Art. no. 1, Jan. 2013, S. Nori et al., ’Grafted human-induced pluripotent stem-cell-derived neurospheres promote motor functional recovery after spinal cord injury in mice’, Proc. Natl. Acad. Sci., vol. 108, no. 40, pp. 16825-16830, Oct. 2011, and A. Yasuda et al., ’Significance of Remyelination by Neural Stem / Progenitor Cells Transplanted into the Injured Spinal Cord’, STEM CELLS, vol. 29, no. 12, pp. 1983-1994, 2011.

[0067] For SCI-related iPSC cell therapy, neural progenitor and stem cells (NPCs / NSCs), such as spinal cord progenitor cells (SCPCs), are differentiated from various established iPSC cell lines on Matrigel-coated tissue culture plastic for specific time periods, which vary per protocols incorporated by reference in their entirety. See, for example, S.H.T. Tay, Winanto, Z.J. Khong, Y.H. Koh, and S.Y. Ng, "Generation of Cortical, Dopaminergic, Motor, and Sensory Neurons from Human Pluripotent Stem Cells," in Methods in Molecular Biology. New York, NY: Springer US, 2021, incorporated by reference in its entirety. This process differentiates iPSCs into NPCs, which are then implanted directly into the injured site of the spinal cord or as part of a tissue scaffold. See, for example, JE Shin et al., "Brain and spinal cord injury repair by implantation of human neural progenitor cells seeded onto polymer scaffolds," Exp. Mol. Med., vol. 50, no. 4, p. 39, April 2018, which is incorporated by reference in its entirety. Although the differentiation process of iPSCs into SCPCs is relatively efficient (typically exceeding 80% differentiation efficiency by day 10), the risk of tumorigenicity after transplantation remains a significant concern.See, for example, Q. Qu et al., "High-efficiency motor neuron differentiation from human pluripotent stem cells and the function of Islet-1," Nat. Commun., vol. 5, no. 1, Art. no. 1, Mar. 2014; Z.-W. Du et al., "Generation and expansion of highly pure motor neuron progenitors from human pluripotent stem cells," Nat. Commun., vol. 6, no. 1, Art. no. 1, Mar. 2015; N. Balafkan et al., "A method for differentiating human induced pluripotent stem cells toward functional cardiomyocytes in 96-well microplates," Sci. Rep., vol. 10, no. 1, Art. no. 1, Oct. 2020; and U. Ben-David and N. Benvenisty, "The tumorigenicity of human embryonic and induced pluripotent stem cells', Nat. Rev. Cancer, vol. 11, no. 4, pp. 268-277, April 2011, and S. Nori et al., 'Long-Term Safety Issues of iPSC-Based Cell Therapy in a Spinal Cord Injury Model: Oncogenic Transformation with Epithelial-Mesenchymal Transition', Stem Cell Rep., vol. 4, no. 3, pp. 360-373, March 2015.Furthermore, even though the use of some iPSC lines has been confirmed and validated as safe, variations in differentiation efficiency or tumor-like overgrowth remain recurring problems when iPSCs are differentiated into neural progenitor cells and subsequently transplanted into injured spinal cords. See, for example, S. Nori et al., "Long-Term Safety Issues of iPSC-Based Cell Therapy in a Spinal Cord Injury Model: Oncogenic Transformation with Epithelial-Mesenchymal Transition," Stem Cell Rep., vol. 4, no. 3, pp. 360-373, Mar. 2015, incorporated by reference in its entirety. The small number of remaining undifferentiated iPSC-like cells poses a significant safety risk due to their potential neoplastic nature in transplanted tissues. See, for example, J. Carlos and AL, 'Safety Assessment of Reprogrammed Cells Prior to Clinical Applications: Potential Approaches to Eliminate Teratoma Formation', in Pluripotent Stem Cells, D. Bhartiya, Ed., InTech, 2013, which is incorporated herein by reference in its entirety.

[0068] Previously, measurements of micromagnetic resonance relaxometry (MRR) have been used as a label-free assay to detect malaria infection, oxidative stress in the blood, and most recently, senescence in MSCs. See, for example, WK Peng et al., 'Micromagnetic resonance relaxometry for rapid label-free malaria diagnosis', Nat. Med., vol. 20, no. 9, pp. 1069-1073, Sep. 2014; WK Peng, L. Chen, BO Boehm, J. Han, and TP Loh, 'Molecular phenotyping of oxidative stress in diabetes mellitus with point-of-care NMR system', Npj Aging Mech. Dis., vol. 6, no. 1, Art. no. 1, Oct. 2020; and SSThamarath et al., 'Rapid and Live-cell Detection of Senescence in Mesenchymal Stem Cells by Micro Magnetic Resonance Relaxometry', Biophysics, preprint, Jun. 2022, each of which is incorporated by reference in its entirety. Measurement of spin-spin relaxation time (T2) is sensitive to the paramagnetic content of the sample, which in the context of cell biology is the presence of iron (Fe 3+) correlates with the intracellular content of ATP. See, for example, M.S. Petronek et al., "Quantum chemical insight into the effects of the local electron environment on T2*-based MRI," Sci. Rep., vol. 11, no. 1, p. 20817, October 2021, incorporated herein by reference in its entirety. This study reports the novel use of MRR for rapid endpoint analysis of iPSC and SCPC phenotypes closely related to safety and quality parameters. Thus, MRR holds great potential for determining critical quality attributes (CQAs) in iPSC-derived progeny and is ideally suited as a quality control tool in cell therapy manufacturing.

[0069] T2 measurement with magnetic resonance relaxometry (MRR) to assess variations in iPSC quality on day 1 The sample preparation process is shown in both Figures 4 and 5. The method shown in Figure 4 uses suspended cells, while Figure 5 shows the method for a pellet of cells. Note that higher magnetic content results in shorter T2 relaxation times.

[0070] Batch-to-batch T2 relaxation times of induced pluripotent stem cells (iPSCs) show cellular variability Typically, existing protocols grow iPSCs by periodic passaging. Each new passage is considered the same as the previous passage. Over the past year, several magnetic measurements have been performed on iPSCs at each passage over an extended period (+6 months). As shown in Figure 6, these measurements demonstrate that, contrary to current opinion, laboratory-cultured iPSCs do not exhibit Fe 3+ Thus, iPSCs at one passage cannot be considered the same as those at the previous passage, or perhaps even the same as when they were first extracted.

[0071] T2 relaxation times under controlled single cell passaging conditions Next, to further understand some of the factors that may lead to such variability, we conducted a one-month study using the MRR method, controlling parameters such as seeding density, passaging time, and plate type passage in single cells. The T2 relaxation times are shown in Figure 7, which, similar to Figure 6, demonstrates the variation in intracellular iron levels based on T2 relaxation time. Therefore, controlling such variation in iPSC production is difficult, highlighting the importance of critical quality attributes (CQAs) for such quality variation (e.g., T2) in cell manufacturing.

[0072] During this experiment, the number of cells harvested was also recorded. The data, shown in Figure 8, demonstrate that the T2 relaxation time per passage correlates with the number of cells harvested at the next passage. This suggests that the iron biology of the cells most certainly influences their growth.

[0073] Relating T2 relaxation times to population doubling times As shown in Figure 6, it was clear that iPSC growth was affected by their iron biology. As another measure of their growth, we performed similar tests using cells passaged via an alternative method, via clumps. Because single-cell passaging is known to cause genetic abnormalities in iPSCs, propagation of iPSCs via clumps is very common. See, for example, Garitaonandia, I., et al., 2015. Increased risk of genetic and epigenetic instability in human embryonic stem cells associated with specific culture conditions. PloS One, 2015, 10(2), p. e0118307, which is incorporated by reference in its entirety. iPSC growth was measured by population doubling time, obtained from computer analysis of culture confluency from microscopic images. Population data time versus T2 relaxation time is plotted in Figure 9. MRR instruments can determine the population doubling time of cultures.

[0074] Effect of freeze-thaw protocols on iPSCs and T2 relaxation time Finally, aside from cell passaging, another common laboratory technique for preserving cells is cryopreservation. While necessary for cell production, such a freeze-thaw process can significantly affect cell phenotype and quality. Data for frozen and thawed iPSCs versus uncryopreserved iPSCs are shown in Figure 10. These data demonstrate that the freeze-thaw process significantly impacts cells with respect to their iron phenotype.

[0075] Large clumps during seeding slow population doubling times When cells are seeded as clumps, it is possible to control the number of clumps seeded, but not the size of the clumps. It is generally known that if the clumps are too small, the cells within the clumps are likely to die. However, if the clumps are large, controlling this distribution can be difficult. The clump size was also determined in conjunction with the T2 relaxation time, culture density, and population doubling time measurements from Figure 9. In Figure 11, as the proportion of cells larger than 80 μm increases, the population doubling time also increases. This may be important because a rapid population doubling time may be associated with healthier iPSCs. See, for example, Ruiz, S., et al., A high proliferation rate is required for cell reprogramming and maintenance of human embryonic stem cell identity. Current Biology, 2011, 21(1), pp. 45-52, which is incorporated by reference in its entirety.

[0076] Based on the data in Figures 9 and 11, this may be an uncontrolled factor due to clump size, which affects variability in iPSC cell culture. Whether cell culture is controllable through clump size or not, MRR can measure their variable passage up to passage in a label-free, non-destructive manner. See, for example, Sullivan, S., et al., "Quality control guidelines for clinical-grade human induced pluripotent stem cell lines." Regenerative medicine, 2018, 13(7), pp. 859-866, which is incorporated by reference in its entirety.

[0077] Correlation between CQA measurements on day 1 and SCPC quality variation on day 10 Spinal cord progenitor cells (SCPCs) are neural precursor cells derived from induced pluripotent stem cells (iPSCs). However, during this differentiation process, there was a great deal of variability in the differentiation outcome, especially in the residual level of OCT4 expression. To characterize these "impurity" levels, a correlation was found between the T2 relaxation time values ​​of the MRR of these SCPCs (at day 10) and their residual OCT4 expression. Various experiments were performed to study the differentiation outcome of SCPCs at different points in the process.

[0078] As shown in Figure 12 below, the T2 values ​​of SCPCs at day 10 correlate with the percentage of OCT4-positive cells in the culture. Because OCT4+ cells represent cells with restricted differentiation, the level of OCT4+ provides a clear quality metric for the SCPCs produced at day 10, immediately prior to neural tissue construction and implantation. Therefore, label-free rapid analysis of T2 levels of SCPCs at day 10 can be used as a valid CQA measure to assess the risk of cancerous tissue development in implants.

[0079] Importantly, the general quality of iPSCs at day 1, as quantified by MRR T2 measurements, can be correlated with the quality (percentage of OCT4+ cells) of SCPCs at day 10 (see Figure 13). This finding can be considered an important step toward truly actionable CQA measurements, which can be implemented as early as day 1 to predict the ultimate likelihood of successful proliferation and differentiation into target SCPCs and neural stem cells. Given that many known and unknown factors in iPSC proliferation lead to significant variability, identifying good and bad batches of cells as early as possible can be meaningful for the overall cell production and manufacturing workflow.

[0080] Other experiments were also carried out. T2 increased as iPSCs differentiated into SCPCs over 10 days.

[0081] SCPCs were generated from iPSCs using three small molecules that mimic the innate developmental pathway of the spinal cord. See, for example, H. Kumamaru et al., "Generation and post-injury integration of human spinal cord neural stem cells," Nat. Methods, vol. 15, no. 9, Art. no. 9, September 2018, and K. Kajikawa et al., "Cell therapy for spinal cord injury by using human iPSC-derived region-specific neural progenitor cells," Mol. Brain, vol. 13, no. 1, p. 120, December 2020, each of which is incorporated by reference in its entirety. Quantification of cell phenotype during differentiation is typically obtained by FACS or immunocytochemical staining. Therefore, the differentiation efficiency of SCPCs on day 10 was quantified by the expression of pluripotency marker (OCT4) and neural progenitor marker (SOX1).

[0082] Changes in OCT4 and SOX1 expression were observed as iPSCs differentiated into SCPCs over three different time points, including day 1 (undifferentiated iPSCs), day 4 (midpoint iPSCs), and day 10 (SCPCs). Figure 14A shows the results of immunostaining for OCT4 and SOX1 at days 1, 4, and 10. As iPSCs began to differentiate into SCPCs, high SOX1 expression was observed on day 4, while OCT4 expression was still decreased. On the final day of differentiation, SOX1 expression was still highly expressed, and OCT4 expression was relatively low. This small cell population that expressed OCT4 was typically found to be remaining undifferentiated iPSCs (marked by an open circle in the merged image at day 10).

[0083] A significant increase in T2 (p<0.0001) was observed when iPSCs were differentiated into SCPCs over 10 days (Fig. 14B). + Quantification of cells was performed using iPSCs differentiated into SCPCs after 10 days using OCT4 + To demonstrate the changes in T2 during differentiation of iPSCs into SCPCs over a 10-day period, T2 was measured with OCT4. + The results were plotted against the cells (Fig. 14D).

[0084] Fe quantified by FACS 3+ Staining revealed that the T2 values ​​of iPSCs and SCPCs were significantly higher than their intracellular Fe 3+ The results confirmed that the RPE fluorescence intensity corresponded to the content of iPSCs (Figure 14E). iPSCs exhibited a higher RPE fluorescence mean intensity (i.e., a higher Fe content) compared to SCPCs. 3+ It was shown that it has a high content of hydroxybenzoates. The presence of artificially spiked iPSCs in the SCPC population reduced the measured T2 values ​​in a dose-dependent manner, representing a safety risk.

[0085] To verify that the T2 values ​​of SCPCs decrease with increasing iPSC presence, SCPCs were artificially spiked with 1-10% iPSCs. Correspondingly, a significant decrease in T2 (p<0.0001) was observed when the spike ratio increased to 5% and above (Figure 15A).

[0086] To assess the potential safety risk associated with lower T2 SCPC cultures, spiked samples were subjected to colony formation assays. Specifically, by culturing all experimental groups in iPSC maintenance medium for 6 days, an increase in iPSC colonies and colony size was observed with increasing amounts of spiked iPSCs (Figures 15B-15C).

[0087] OCT4 levels in SCPC populations at day 10 correlate with T2 measurements at day 10 and can be predicted by T2 measurements of iPSCs at initiation To investigate whether MRR could detect mutations in remaining undifferentiated iPSCs at day 10, we compared T2 at day 10 with OCT4 at day 10. + The results showed that an increase in the number of OCT4-positive cells in SCPCs on day 10 decreased T2 measurements in a negative linear correlation trend line (FIG. 16A).

[0088] To determine whether T2 on day 1 could provide a prediction of differentiation outcome on day 10, several correlations were performed. Specifically, T2 measurements on day 1 were correlated with T2 measurements on day 10 and OCT4 on day 10. + The correlation was that lower T2 in day 1 iPSCs correlated with lower T2 in day 10 SCPCs and higher OCT4 in day 10. + The results clearly showed that the levels of α-glucan were significantly increased (Figures 16B to 16C). T2 decreases in line with the decline in levels of neural stem cell and progenitor cell markers.

[0089] To evaluate the effectiveness of MRR in reflecting SCPC quality, the degree of SCPC differentiation was adjusted by decreasing the concentration of CHIR-99021 in the neural differentiation medium. CHIR-99021 is a potent GSK3 inhibitor involved in the activation level of the Wnt signaling pathway, which controls the differentiation efficiency of iPSCs into various progenitor cell types. See, for example, Z.-W. Du et al., "Generation and expansion of highly pure motor neuron progenitors from human pluripotent stem cells," Nat. Commun., vol. 6, no. 1, Art. no. 1, March 2015, and T. Qian, T. M. Heaster, A. R. Houghtaling, K. Sun, K. Samimi, and M. C. Skala, "Label-free imaging for quality control of cardiomyocyte differentiation," Nat. Commun., vol. 12, no. 1, p. 4580, December 2021, each of which is incorporated by reference in its entirety.

[0090] Experimental groups included no CHIR-99021 (CHIR 0, 0 μM), a tenth lower concentration of CHIR-99021 (CHIR 1 / 10, 0.425 μM), and a control group (CHIR normal, 4.25 μM). The decrease in CHIR concentration was associated with SOX1 upregulation in SCPCs at day 10. + , Nestin + (neural stem cell marker) and OCT4 + It was shown to affect the cellular level (Figures 17A-C).

[0091] More importantly, T2 was significantly lower in CHIR0 (p<0.01) and CHIR1 / 10 (p<0.0001) compared with CHIR normal (Figure 17D). T2 also corresponded to decreased expression of SOX1 and Nestin and increased expression of OCT4 in both CHIR0 and CHIR1 / 10 compared with CHIR normal (Figures 17E-17G).

[0092] Validation in different iPS cell lines, CLEC23 and BJ iPSC To confirm and validate the robustness of the MRR in identifying the differentiation efficiency of iPSCs into SCPCs, we verified our initial findings using another iPSC line. Specifically, the use of the BJ-iPSC cell line represented fibroblast-derived iPSCs previously used to generate spinal motor neurons. See, for example, S.-Y. Ng et al., "Genome-wide RNA-Seq of Human Motor Neurons Implicates Selective ER Stress Activation in Spinal Muscular Atrophy," Cell Stem Cell, vol. 17, no. 5, pp. 569-584, November 2015, and J.-H. Hor et al., "ALS Motor Neurons Exhibit Hallmark Metabolic Defects that Are Rescued by SIRT3 Activation," Cell Death Differ., vol. 28, no. 4, Art. no. 4, April 2021, both of which are incorporated by reference in their entireties.

[0093] The T2 and OCT4 levels of BJ-iPSCs were in a similar range to those of CLEC23 iPSCs (Figure 18A). However, T2 of BJ-SCPCs at day 10 was generally higher compared to CLEC23 SCPCs at day 10 (Figures 18B-18C). T2 of BJ-SCPCs at day 10 corresponded to higher SOX1 and lower OCT4 expression, respectively, compared to CLEC23 SCPCs.

[0094] Detecting cells with tumorigenic potential and ensuring the quality of progenitor cells used in regenerative cell therapy remain significant obstacles. For example, a recent clinical trial of iPSC-derived islet beta cell therapy for diabetes resulted in teratomas two months after transplantation. See, for example, L. Han et al., "Distinctive Clinical and Pathologic Features of Immature Teratomas Arising from Induced Pluripotent Stem Cell-Derived Beta Cell Injection in a Diabetes Patient," Stem Cells Dev., vol. 31, no. 5-6, pp. 97-101, Mar. 2022, incorporated by reference in its entirety. Furthermore, the tumors were characterized as immature teratomas and expressed both OCT4 and SOX2 in immunofluorescence assays. Nori and colleagues attempted to transplant iPSC-derived neural progenitor cells into a mouse model of SCI, and found early functional recovery and synaptogenesis 47 days after transplantation. See, for example, S. Nori et al., "Long-Term Safety Issues of iPSC-Based Cell Therapy in a Spinal Cord Injury Model: Oncogenic Transformation with Epithelial-Mesenchymal Transition," Stem Cell Rep., vol. 4, no. 3, pp. 360-373, March 2015, which is incorporated by reference in its entirety. However, when the study was extended beyond 103 days, tumor formation was observed, leading to a decline in motor function. In these tumors, the OCT4 transgene was found to be activated, and anaplastic Nestin (AN) was expressed. +Neurons were prominently present. Recent clinical trials of transplanting iPSC-derived neural stem and progenitor cells also revealed the presence of residual undifferentiated iPSCs by quantifying OCT4 expression in the cell population. See, for example, K. Sugai et al., "First-in-human clinical trial of transplantation of iPSC-derived NS / PCs in subacute complete spinal cord injury: Study protocol," Regen. Ther., vol. 18, pp. 321-333, December 2021, which is incorporated by reference in its entirety. Thus, OCT4 expression in iPSC-derived progeny was not observed. + Detecting the cells is important to ensure and validate the safety of iPSC-related cell therapy products.

[0095] Current assays for quantifying the phenotype of iPSCs and progenitor cells use label-based fluorescent markers that are analyzed by flow cytometer or visualized by fluorescence microscopy. See, for example, T. Kuroda et al., "Highly Sensitive In Vitro Methods for Detection of Residual Undifferentiated Cells in Retinal Pigment Epithelial Cells Derived from Human iPS Cells," PLOS ONE, vol. 7, no. 5, p. e37342, May 2012, incorporated by reference in its entirety. However, these assays often disturb or destroy cells after terminal fixation, rendering them unusable for subsequent applications. Furthermore, these assays are expensive, laborious, and time-consuming, ultimately inadequate for quality control of iPSCs or iPSC-derived progeny in biological production processes. Biochemical and omic-based methods are the ultimate standard for determining cellular phenotypic characteristics. Yet, most of them are population-based assays, requiring averaging of cell signaling across a population of cells. Given the general heterogeneity of stem cells and their progenitors, they are generally not sufficient to monitor the presence of low-abundance cells that pose a safety risk.

[0096] On the other hand, label-free techniques, which can characterize cellular biophysical properties that correlate with cell quality and safety, allow for rapid, non-destructive assessment of the quality of cell batches, making them ideally suited for large-scale cell manufacturing. In this study, we describe a rapid, non-destructive method for validating the safety and quality phenotype of in vitro-generated iPSC-derived SCPCs.

[0097] The MRR-mediated T2 readout was shown to correspond to the phenotypic transition from iPSCs to SCPCs, with iPSCs generally exhibiting greater paramagnetic content (higher intracellular Fe 3+ ) (Fig. 14A to Fig. 14E).3+ The staining also confirmed that T2 measurements of iPSCs and SCPCs 3+ These findings demonstrate that T2 measurements are correlated with intracellular Fe levels (Fig. 14E). 3+ These results clearly demonstrate that OCT4 is a good surrogate for label-free measurement of iron content. The difference in iron content between iPSCs and SCPCs was essential for developing the hypothesis of detecting the purity of SCPC populations after their differentiation process. Therefore, OCT4 levels in SCPCs, which can be measured via the MRR system, were investigated. This measurement allows for an evaluation of the differentiation efficiency endpoint between SCPC batches.

[0098] Spiking iPSCs demonstrated that various proportions of iPSCs could be estimated using the MRR system (Figure 15A). Further verification of the safety risk in SCPC associated with lower T2 values ​​(due to the increased presence of residual undifferentiated iPSCs) was performed by colony culture assay. Colony formation assay was previously used to evaluate and determine the presence of any residual undifferentiated iPSCs in differentiated cells. See, for example, K. Tano, S. Yasuda, T. Kuroda, H. Saito, A. Umezawa, and Y. Sato, "A Novel In Vitro Method for Detecting Undifferentiated Human Pluripotent Stem Cells as Impurities in Cell Therapy Products Using a Highly Efficient Culture System," PLOS ONE, vol. 9, no. 10, pp. e110496, October 2014, and T. Watanabe et al., "Multisite studies for validation and improvement of a highly efficient culture assay for detection of undifferentiated human pluripotent stem cells intermingled in cell therapy products," Cytotherapy, vol. 23, no. 2, pp. 176-183, February 2021, each of which is incorporated by reference in its entirety. However, this study used this assay to illustrate safety issues associated with low T2 values. The results of the spike-in experiments showed that increasing the amount of iPSCs in the SCPC population resulted in a proportional decrease in T2, but these results were insufficient to conclude that MRR can be used to detect residual undifferentiated iPSCs in the actual day 10 SCPC population.First, the T2 of iPSCs consisted of significant batch-to-batch variability, causing some spike-in experiments to have a larger decrease in T2 compared to others. Second, it was possible that the remaining undifferentiated iPSCs would not exhibit the same T2 as the undifferentiated iPSCs at day 1. Therefore, we looked more closely at the SCPCs at day 10 and performed several different correlations to demonstrate the ability of MRR to identify variations in the remaining undifferentiated iPSCs.

[0099] Several batches of day 10 SCPCs of CLEC23 were evaluated for their number of remaining undifferentiated iPSCs by immunofluorescence staining, and we found that T2 in day 10 SCPCs was significantly higher than that in OCT4. + The T2 values ​​correlated with the percentage of remaining undifferentiated iPSCs (Figure 16A). This phenomenon occurred because SCPC populations with a higher number of remaining undifferentiated iPSCs effectively contributed to the total iron content present in the sample. This effectively lowered the T2 values ​​compared to SCPCs with fewer remaining undifferentiated iPSCs. Furthermore, iPSCs with lower T2 values ​​exhibited lower T2 and higher OCT4 levels after differentiation into SCPCs (Figures 16B-C). This indicates that T2 measurements of iPSCs as early as day 1 in the differentiation process can be used to predict the resulting efficiency of iPSC-to-SCPC differentiation. This predictive ability is highly beneficial for determining whether a batch of iPSCs will yield a high-quality batch of SCPCs. Such an early-stage quality assessment tool would be highly desirable in iPSC manufacturing workflows, which can suffer from significant donor-to-donor and batch-to-batch quality variation.

[0100] Although the absence of undifferentiated iPSCs in the SCPC population measured by MRR can serve as a safety CQA, it does not automatically translate into high-quality SCPCs. To establish the feasibility of MRR as a quality indicator, we performed negative inhibition of differentiation efficiency to obtain T2 of poorly differentiated SCPCs. Inhibition of differentiation by decreasing CHIR concentration resulted in lower SOX1 expression. + and Nestin +Cells and higher OCT4 + Based on previous findings, OCT4 + This strongly suggests that the increased presence of cells also contributed to the decreased T2 of poorly differentiated SCPCs. These findings demonstrated that T2 measurements strongly correlate with the outcome of poor SCPC differentiation (Figures 17D-17G). These results again confirm that T2 of differentiated SCPC populations can indicate whether the differentiation was of poor quality.

[0101] Using another iPSC line (BJ-iPSC), we demonstrated that the T2 values ​​of iPSC and SCPC were not innate to the CLEC23 iPSC line. Differences existed in the outcome of differentiation of CLEC23 and BJ iPSC into SCPC. SOX1 expression was significantly higher in BJ-SCPC at day 10 than in CLEC23 SCPC at day 10. + A higher proportion of cells were OCT4 + The percentage of cells was found to be lower in each group. Corresponding to the phenotype, the T2 measurements of BJ-SCPCs on day 10 were also found to be higher than those of CLEC23-SCPCs (Figures 18B-18C). This strongly suggests that higher T2 values ​​correspond to higher differentiation efficiencies of iPSCs to SCPCs. This indicates that T2 can be used to evaluate the differentiation potential of other iPSC lines in differentiation to SCPCs.

[0102] Currently, the precise mechanistic relationship between intracellular iron levels and cell phenotype (e.g., stem cells, progenitors, and fully differentiated cells) is poorly understood, despite the recent explosion of scientific interest in this topic. Previously, magnetic resonance imaging (MRI) has been reported to track in vitro differentiation of MSCs into neurons by coupling the ferritin gene to a neural cell-specific promoter. See, for example, C. Song et al., "Use of Ferritin Expression, Regulated by Neural Cell-Specific Promoters in Human Adipose Tissue-Derived Mesenchymal Stem Cells, to Monitor Differentiation with Magnetic Resonance Imaging In Vitro," PLOS ONE, vol. 10, no. 7, p. e0132480, July 2015, which is incorporated by reference in its entirety. Furthermore, the differentiation outcomes of iPSCs into various progenitors have been enhanced by iron treatment or the addition of iron-binding proteins. See, for example, D. Lu et al., "Accelerated Neuronal Differentiation Toward Motor Neuron Lineage from Human Embryonic Stem Cell Line (H9)," Tissue Eng. Part C Methods, vol. 21, no. 3, pp. 242-252, Mar. 2015, and F. Zhang et al., "Transferrin improved the generation of cardiomyocytes from human pluripotent stem cells for myocardial infarction repair," J. Mol. Histol., vol. 52, no. 1, pp. 87-99, Feb. 2021, each of which is incorporated by reference in its entirety. However, no studies have elucidated the changes in intracellular iron content during differentiation of one cell type into another.Furthermore, our data demonstrate that T2 of MRR is a non-destructive, label-free endpoint assay to rapidly measure the phenotypic differentiation efficiency of iPSC-derived neural stem and progenitor cells for rapid validation of safety and quality parameters.

[0103] This is because intracellular Fe 3+ This is the first reported study to identify differences in cell types through their content. However, the use of label-free techniques to measure or analyze cell phenotypes has previously been demonstrated by Qian et al., who implemented an autofluorescence system to quantify the differentiation efficiency of iPSC-derived cardiac progenitor cells. See, for example, T. Qian, T.M. Heaster, A.R. Houghtaling, K. Sun, K. Samimi, and M.C. Skala, "Label-free imaging for quality control of cardiomyocyte differentiation," Nat. Commun., vol. 12, no. 1, p. 4580, December 2021, each of which is incorporated by reference in its entirety. Autofluorescence from endogenous fluorophores, such as NADH and FAD, is involved in the general energy metabolism of cells, suggesting that stem cells and their progenitors have distinct energy phenotypes. See, for example, D. Lu et al., 'Accelerated Neuronal Differentiation Toward Motor Neuron Lineage from Human Embryonic Stem Cell Line (H9)', Tissue Eng. Part C Methods, vol. 21, no. 3, pp. 242-252, Mar. 2015, which is incorporated by reference in its entirety. 2+ ) but the intracellular Fe associated with "storage iron" 3+ Quantitation of paramagnetic ions was determined to be an indicator of general cellular metabolic activity.

[0104] Cellular iron content has recently attracted considerable interest among researchers due to its potential implications in many physiological and pathophysiological processes, including aging, activation, cellular senescence, cancer, and inflammation. For example, WJChen, GPKung, and JPGnana-Prakasam, 'Role of Iron in Aging Related Diseases', Antioxidants, vol. 11, no. 5, p. 865, Apr. 2022, T. Sato, JSShapiro, H.-C. Chang, RAMiller, and H. Ardehali, 'Aging is associated with increased brain iron through cortex-derived hepcidin. expression',eLife,vol.11,p.e73456,Jan.2022,S.Kuvibidila,RPWarrier,and B.Surendra Baliga,'An overview of the role of iron in T cell activation',J.Trace ElemExp.Med.,vol.16,no.4,pp.219-225,2003,S.Ni,Y.Yuan,Y.Kuang,and X.Li,'Iron Metabolism and Immune Regulation',Front.Immunol.,vol.13,2022,Accessed:Jun.12,2023,DWKillilea,SLWong,HSCahaya,H.Atamna,and BNAmes,'Iron Accumulation during Cellular Senescence',Ann.NYAcad.Sci.,vol.1019,no.1,pp.365-367,Jun.2004, A.Cozzi et al.,'Stem Cell Modeling of Neuroferritinopathy Reveals Iron as a Determinant of Senescence and Ferroptosis during Neuronal Aging',Stem Cell Rep.,vol.13,no.5,pp.832-846,Nov.2019,D.H. Manz, NLBlanchette, BT Paul, FMTorti, and SVTorti, 'Iron and cancer: recent insights', Ann.NYAcad.Sci., vol. 1368, no. 1, pp. 149-161, Mar. 2016, Q. Guo et al., 'The Role of Iron in Cancer Progression',Front.Oncol.,vol.11,2021,Accessed:Jun.12,2023, T.Basak and RKKanwar,'Iron imbalance in cancer:Intersection of deficiency and overload',Cancer Med.,vol.11,no.20,pp.3837-3853,2022,M.Wessling-Resnick,'Iron Homeostasis and the Inflammatory Response', Annu. Rev. Nutr., vol. 30, pp. 105-122, August 2010; D.B. Kell and E. Pretorius, 'Serum ferritin is an important inflammatory disease marker, as it is mainly a leakage product from damaged cells', Metallomics, vol. 6, no. 4, pp. 748-773, April 2014; and O. Marques, G. Weiss, and M.U. Muckenthaler, 'The role of iron in chronic inflammatory diseases: from mechanisms to treatment options in anemia of inflammation', Blood, vol. 140, no. 19, pp. 2011-2023, November 2022, each of which is incorporated by reference in its entirety. However, Fe in these important biological contexts. 2+ / Fe3+ There is no systematic understanding of the exact role of Fe. 2+ and Fe 3+ This is exacerbated by the difficulty in obtaining accurate measurements of Fe. 2+ , Fe 3+Conventional assays and methods for detecting iron, and even total iron concentrations, are largely inaccurate due to the nature of the iron ion, inaccurate correlations, and limitations of current technology. For example, T. Hirayama and H. Nagasawa, 'Chemical tools for detecting Fe ions', J. Clin. “labile iron pool” or only a fraction of it?',Biochem.J.,vol.403,no.Pt 2,pp.261-266,Apr.2007,U.Abbasi,S.Abbina,A.Gill,V.Bhagat,and JNKizhakkedatu,'A facile colorimetric method for the quantification of labile iron pool and total iron in cells and tissue specimens',Sci.Rep.,vol.11,p.6008,Mar.2021,JSRohrer,MSJoo,E.Dartyge,DESayers,A.Fontaine,and ECTheil,'Stabilization of iron in a ferrous form by ferritin.A study using dispersive and conventional x-ray absorption spectroscopy.',J.Biol.Chem.,vol.262,no.28,pp.13385-13387,Oct.1987,MAKnovich,JAStorey,LGCoffman,and SVTorti,'Ferritin for the Clinician',Blood Rev.,vol.23,no.3,pp.95-104,May 2009, V. Fiorito, S. Geninatti Crich, L.Silengo,F.Altruda,S.Aime,and E.Tolosano,'Assessment of iron absorption in mice by ICP-MS measurements of(57)Fe levels',Eur.J.Nutr.,vol.51,no.7,pp.783-789,Oct.2012,B.Ari,SZCan,and S.Bakirdere,'Traceable and accurate quantification of iron in seawater using isotope dilution calibration strategies by triple quadrupole ICP-MS / MS:Characterization measurements of iron in a candidate seawater CRM',Talanta,vol.209,p.120503,Mar.2020 and C.Song et al.,'Use of Ferritin Expression,Regulated by Neural Cell-Specific Promoters in Human Adipose Tissue-Derived Mesenchymal Stem Cells,to Monitor Differentiation with Magnetic Resonance Imaging In Vitro', PLOS See, ONE, vol. 10, no. 7, p. e0132480, July 2015, each of which is incorporated by reference in its entirety. Furthermore, these methods typically require additional reagents or kits to measure iron, which ultimately exposes the biological sample to further chemical or biological treatment.

[0105] The MRR system measures T2 in living cells, providing a unique tool for cellular iron biology. T2 measurements of iPSCs and SCPCs in this study correlated well with markers (e.g., OCT4, SOX1, and Nestin) that require terminal fixation for analysis. Furthermore, this method allows for the analysis of small numbers of cells (<2 × 10 5) and does not require any chemical or biological treatment, allowing the same cells to be subjected to subsequent biological or functional measurements that can be compared or correlated with their T2 values. This was demonstrated in a previous study that used MRR to demonstrate the quality of MSCs by quantifying their level of senescence, which directly impacts their downstream therapeutic efficacy. See, for example, S.T. Hamarath et al., "Rapid and Live-Cell Detection of Senescence in Mesenchymal Stem Cells by Micro Magnetic Resonance Relaxometry," Stem Cells Transl. Med., p. szad014, Mar. 2023, incorporated by reference in its entirety.

[0106] It is important to note that MRR is not without limitations: although MRR has been reported to be an indicator of T2-mediated quality, cellular phenotype does not necessarily correlate directly with subsequent downstream function.

[0107] It is also important to note that variations in differentiation efficiency may possibly be due to the initial quality of iPSCs. Recent publications suggest that iron overload or underload in iPSCs can severely affect the maintenance of their pluripotency. See, for example, Z. Han et al., "Iron Homeostasis Determines the Fate of Human Pluripotent Stem Cells Via Glycerophospholipids-Epigenetic Circuit," Stem Cells, vol. 37, no. 4, pp. 489-503, April 2019, and Z. Han et al., "Iron Overload Inhibits Self-Renewal of Human Pluripotent Stem Cells via DNA Damage and Generation of Reactive Oxygen Species," FEBS Open Bio, vol. 10, no. 5, pp. 726-733, May 2020, each of which is incorporated by reference in its entirety. Therefore, this recent finding presents the tremendous potential of MRR to clarify further insights into iron homeostasis in iPSCs for the purpose of quality control. Furthermore, recent data indicate that mechanical stress, such as vibration, has a profound downstream impact on iPSC maintenance. See, for example, K. Kanie et al., "Effect of mechanical vibration stress in cell culture on human induced pluripotent stem cells," Regen. Ther., vol. 12, pp. 27-35, December 2019, which is incorporated herein by reference in its entirety. Another future direction for MRR is to investigate the role of Fe 3+ The goal is to combine measurements with other label-free techniques to perform multivariate analyses of cell quality. MRR provides information about cell quality through a single biophysical property, allowing for comparison with other biophysical factors such as cell size, impedance, and autofluorescence. 3+Combining measurements provides a deeper and more robust analysis of cell quality, and the inventors believe that such multivariate analysis will be a major benefit and game changer for the cell therapy manufacturing industry.

[0108] Intracellular Fe to quantify iPSC and SCPC phenotypes 3+ Using this technology, the ability of the label-free MRR technique to perform rapid, non-destructive endpoint assessment of the safety and quality of SCPCs has been demonstrated. T2 measurements have been shown to correlate well with traditional biochemical assays for iPSC and SCPC phenotyping. This indicates that T2 measurements via the MRR system are ideally suited to monitor and assess the safety and quality of iPSC-derived SCPCs generated for application in cell therapy for spinal cord injury.

[0109] The methods and results of the present disclosure are described below.

[0110] material and method Measurement with MRR The MRR consists of a portable permanent magnet with B = 0.5 T (Metrolab Instruments, Plan-les-Ouates, Switzerland) and a benchtop NMR console (Kea Magritek, Wellington, New Zealand). Inside the magnet, at a resonant frequency of 21.65 MHz, 1 H MRR measurements were performed using a single resonant proton MRR probe with a 900 μm inner diameter detection microcoil. The MRR sample was housed in a microcapillary tube (1,500 μm outer diameter, 950 μm inner diameter) (Fisherbrand, Waltham, MA, USA, 22-260-950). The MRR probe electronics and coil were mounted on a single printed circuit board (Figure 1A). All experiments were performed at 26.3 °C inside the magnet, maintained by a temperature controller (RS Components, UK).

[0111] For all MRR experiments, unless otherwise specified, a normalized cell concentration (6 x 104 cells in a volume of 4 µL) can be used. Cell samples can be centrifuged at 400 g for 5 min, and the supernatant aspirated. The pellet can be suspended in PBS at a concentration of 1.5 x 104 cells / µL, and 4 µL of the suspension was filled into a 4 mm length of microcapillary tubing. The microcapillary tubing can be sealed with a crystal (Leica Microsystems) and attached to a coil for MRR measurements. Proton transverse relaxation time (T2) can be measured by a standard Carr-Purcell-Meiboom-Gill (CPMG) pulse program (Figures 3A-3B). The transmitter power output can be maintained at 12.5 mW for a single 90° pulse with a pulse length of 6 µs for all T2 measurements. A CPMG train of pulses with 4,000 echoes and a 200 µs inter-echo time can be used in all experiments. A recirculation delay of 2 seconds may be used, sufficient for all spins to return to thermal equilibrium. For example, 24 scans may be performed for a signal averaging experiment.

[0112] iPSC culture Two human iPSC lines were routinely cultured and maintained in this study on Matrigel (83.3 μg / mL, Corning, USA)-coated tissue culture plastic dishes in StemMACS™ iPS-Brew XF (Miltenyi Biotech, Germany). (1) iPSCs derived from healthy umbilical cord lining epithelial cells (CLEC23) were kindly provided by Dr. Kah-Leong Lim and CellResearch Corporation Pte Ltd. See, for example, Y. Zhou et al., "Characterization of Human Umbilical Cord Lining-Derived Epithelial Cells and Transplantation Potential," Cell Transplant., vol. 20, no. 11-12, pp. 1827-1841, December 2011, each of which is incorporated by reference in its entirety. CLEC-derived iPSCs were hypothesized to exhibit immune-privileged properties similar to those of CLECs. See, for example, R. Saleh and HM Reza, "Short review on human umbilical cord lining epithelial cells and their potential clinical applications," Stem Cell Res. Ther., vol. 8, no. 1, p. 222, October 2017, and RHG Lim, JXK Liew, A. Wee, J. Masilamani, SKY Chang, and TTPhan, "Safety Evaluation of Human Cord-Lining Epithelial Stem Cells Transplantation for Liver Regeneration in a Porcine Model," Cell Transplant., vol. 29, p. 963689719896559, 2020, each of which is incorporated by reference in its entirety. (2) BJ-iPSCs were derived from BJ-fibroblasts using modified mRNA.See, for example, Winanto, Z.J. Khong, B.-S. Soh, Y. Fan, and S.-Y. Ng, "Organoid cultures of MELAS neural cells reveal hyperactive Notch signaling that impacts neurodevelopment," Cell Death Dis., vol. 11, no. 3, Art. no. 3, Mar. 2020, which is incorporated by reference in its entirety. Tissue culture plates were coated with 3 mL of Matrigel at 37°C for a minimum of 15 minutes before passage. Briefly, cells were cultured daily and passaged via ReleSR™ (STEMCELL Technologies, Canada) upon reaching 70-80% confluency.

[0113] SCPC differentiation Spinal cord progenitor cells (SCPCs) were differentiated from the CLEC23-iPSC and BJ-iPSC cell lines. Methods for generating SCPCs have been derived. See, for example, SHTay, Winanto, ZJKhong, YHKoh, and SYNg, "Generation of Cortical, Dopaminergic, Motor, and Sensory Neurons from Human Pluripotent Stem Cells," in Methods in Molecular Biology. New York, NY: Springer US, 2021, which is incorporated by reference in its entirety. Briefly, iPSCs were removed from tissue culture dishes at 70–80% confluency using Accutase (Nacalai Tesque Inc., Japan) and plated at 800,000 cells / well onto Matrigel-coated tissue culture plastic 6-well plates containing neural induction medium (NIM) supplemented with ROCK inhibitor Y-27632 (ROCKi, 5 μM, Miltenyi Biotech) (1 mL of 83.3 μg / mL for a minimum of 15 min at 37 °C). NIM consisted of DMEM / F12 (50%, Thermo Fisher Scientific, Waltham, MA, USA), neural medium (50%, Miltenyi Biotech), NeuroBrew-21 (1x, Miltenyi Biotech), N2 (1x, Miltenyi Biotech), non-essential amino acids (1x, Thermo Fisher Scientific), Glutamax (0.5x, Thermo Fisher Scientific), LDN-193189 (0.5 μM, Miltenyi Biotech), and CHIR-99021 (CHIR, 4.25 μM, Miltenyi Biotech). On day 3, retinoic acid (RA, 10 μM, Sigma, USA) was added to NIM to induce tail formation. On day 4, halved iPSCs were removed with Accutase and cultured at 2.5 × 10 cells / well in NIM containing RA and ROCKi. 6The cells were transferred to Matrigel-coated tissue culture plastic dishes at 10 cells / dish. On day 5, NIM+RA and ROCKi were aspirated and replenished with NIM+RA. From days 6 to 9, cells received daily media changes of NIM+RA. On day 10, differentiated cells were removed from the dishes with Accutase and then characterized in various assays.

[0114] Measuring MRR The instrument was established in a previous study conducted within the same group. See, for example, WK Peng, L. Chen, BO Boehm, J. Han, and TP Loh, 'Molecular phenotyping of oxidative stress in diabetes mellitus with point-of-care NMR system', Npj Aging Mech. Dis., vol. 6, no. 1, Art. no. 1, October 2020; WK Peng et al., 'Micromagnetic resonance relaxometry for rapid label-free malaria diagnosis', Nat. Med., vol. 20, no. 9, pp. 1069-1073, September 2014; and SSThamarath et al., 'Rapid and Live-Cell Detection of Senescence in Mesenchymal Stem Cells by Micro Magnetic Resonance Relaxometry', Stem Cells Transl. Med., p. szad014, March 2023, each of which is incorporated by reference in its entirety. Briefly, the MRR system consists of a portable permanent magnet with B0 = 0.5 T (Metrolab Instruments, Switzerland) and a benchtop nuclear magnetic resonance (NMR) console (Kea Magritek, New Zealand). 1H MRR measurements were performed inside the magnet at a resonant frequency of 21.015 MHz. The MRR sample was loaded into a microcapillary tube (Fisherbrand, USA) attached to a 900 μm inner diameter detection microcoil inside a single resonant proton MRR probe. The electronics and coil are both part of a printed circuit board in the MRR probe (Figure 1). All MRR experiments were performed at a temperature of 26.3 °C inside the magnet, maintained by a temperature control device (RS Components, UK).

[0115] For all MRR experiments, the iPSC and SCPC concentrations used were fixed at 180,000 cells in a 4 μL volume. The iPSC or SCPC samples were spun down at 300 g for 5 minutes and 1,000 rpm for 3 minutes, respectively, after which the supernatant was aspirated. PBS was then added, and the tubes were spun down at 1,500 rpm for 3 minutes. The supernatant was aspirated, and the cell pellet was transferred to an Eppendorf tube. The cells were counted (INCYTO, Korea), and a sample of 45,000 cells / mL in 50 mL of cell solution was prepared. 4 mL of the cell solution was pipetted into a microcapillary tube, sealed with a ClitoSeal (Leica Microsystems), and attached to the MRR system to obtain readouts. Proton transverse relaxation time (T2) measurements were performed using a standard Carr-Purcell-Meiboom-Gill (CPMG) pulse program (Figure 3A). For all experiments, a 500 μs inter-echo time with 4000 echoes was applied to the CPMG pulse train. A 3-second recirculation delay was applied to allow sufficient time for all spins to return to thermal equilibrium. Signal averaging was performed for all MRR experiments, with a total of 24 scans.

[0116] To quantify changes in T2 during this differentiation process, T2 of iPSCs and SCPCs was measured by MRR on days 1, 4, and 10 of the differentiation process.

[0117] Immunofluorescence staining SCPCs were seeded at 80,000 cells / well on Matrigel-coated 96-well plates. Cells were stained with SOX1, HOXB4, and OCT4 antibodies. Briefly, SCPCs were fixed with 4% paraformaldehyde (PFA, Biotium, USA) for 15 minutes. The fixative was removed and washed twice with PBS. The wells to be fixed were then permeabilized with 0.1% Triton-X in PBS for 15 minutes. After permeabilization, blocking buffer (2% bovine serum albumin (BSA), 5% fetal bovine serum) was added and incubated at room temperature for 1 hour. Primary antibodies were then added, and the samples were incubated overnight at 4°C. The primary antibodies used were SOX1 (1:250, Cell Signaling Technology), HOXB4 (1:250, Abcam), and OCT4 (1:500, Santa Cruz Biotechnology). After removing the primary antibodies, the wells were washed twice with PBS. Secondary antibodies with the corresponding host species and DAPI were then added to the wells and incubated for 1 hour at room temperature without light exposure. The secondary antibodies and DAPI used were donkey AlexaFluor 488-conjugated anti-rabbit IgG (1:500, Thermo Fisher Scientific), donkey AlexaFluor 555-conjugated anti-mouse IgG (1:500, Thermo Fisher Scientific), and DAPI (1:1000, Thermo Fisher Scientific). After removing the primary antibody, wells were washed twice with PBS. Stained cells were imaged using a Leica DMi8 microscope and quantified using CellProfiler.

[0118] Flow cytometry analysis Collect the SCPCs in a 15 mL centrifuge tube and separate 5 x 10 6The cells were adjusted to a density of 1000 cells / mL. They were then fixed with 4% PFA for 15 minutes at room temperature. Once fixed, they were centrifuged at 3000 rpm for 5 minutes and washed once with PBS. The cells were then stained by incubating with primary antibodies reconstituted in permeabilization / blocking buffer (0.5% saponin, 1% BSA) at room temperature for a minimum of 2 hours. The primary antibodies used were SOX1 (1:250, Cell Signaling Technology), Nestin (1:200, Abcam), HOXB4 (1:250, Abcam), and OCT4 (1:500, Santa Cruz Biotechnology). After staining, the cells were centrifuged at 3000 rpm for 5 minutes and washed twice with PBS. The cells were then stained with secondary antibodies reconstituted in permeabilization / blocking buffer for 45 minutes at room temperature in the absence of light. The secondary antibodies used were donkey AlexaFluor488-conjugated anti-rabbit IgG (1:500, Thermo Fisher Scientific) and donkey AlexaFluor555-conjugated anti-mouse IgG (1:500, Thermo Fisher Scientific). Finally, the cells were centrifuged at 3000 rpm for 5 minutes, washed once with PBS, and resuspended in 300 μL of PBS. The cells were analyzed using a Cytoflex flow cytometer (Beckman Coulter, USA).

[0119] iPSC spike and colony culture assay iPSCs were spiked into harvested SCPCs at different ratios, including 1%, 5%, and 10%. SCPCs were then counted and a fixed number of cells were removed to account for the addition of iPSCs. Cells were plated in 6-well plates at 1 x 10 7 Cells were seeded at an initial density of 100 cells / well. They were cultured in 2 mL of hiPSC medium for 6 days, with daily medium changes. Cells were fixed, stained with DAPI and OCT4 antibodies, and imaged using a Leica DMi8 microscope. Colony size was analyzed using the ImageJ particle size analyzer.

[0120] Fe in iPSCs and SCPCs 3+ Staining and quantification Intracellular iron (Fe 3+ ) was determined by reversible fluorescent Fe 3+ This was done via a sensor (RPE). See, for example, Y. Wei, Z. Aydin, Y. Zhang, Z. Liu, and M. Guo, "A Turn-on Fluorescent Sensor for Imaging Labile Fe3+ in Live Neuronal Cells at Subcellular Resolution," ChemBioChem, vol. 13, no. 11, pp. 1569-1573, July 2012, which is incorporated by reference in its entirety. 3+ For staining, a stock solution of RPE (1 mM in acetonitrile) was diluted in PBS to a concentration of 20 μM and 3 × 10 5 The suspended cells were collected and centrifuged to remove the supernatant. The cells were then incubated with PBS containing RPE (20 μM) at 37°C for 20 minutes. After incubation, the cells were washed twice and suspended in PBS for measurement of their fluorescence intensity by flow cytometry.

[0121] statistical analysis Statistical analysis was performed using GraphPad Prism 9. All data were presented as mean ± standard deviation (SD). A p-value <0.05 was considered statistically significant, with * = p<0.05; ** = p<0.01; *** = p<0.001; and **** = p<0.0001.

[0122] The details of one or more embodiments are set forth in the accompanying drawings and description. Other features, objects, and advantages will become apparent from the description, drawings, and claims. While several embodiments of the invention have been described, it will be understood that various modifications can be made without departing from the spirit and scope of the invention. It should also be understood that the accompanying drawings are not necessarily to scale, and that they present somewhat simplified representations of various features and underlying principles of the invention.

Claims

1. A method for evaluating induced pluripotent stem cells and cells differentiated therefrom, comprising: loading a sample containing a plurality of induced pluripotent stem cells and cells differentiated therefrom at a sensor; placing the sample-containing sensor within or near a detection coil of a magnetic resonance relaxometry device; T of the sample ２ Determining the value, and Said T ２ and evaluating the quality of the induced pluripotent stem cells and cells differentiated therefrom from the values ​​obtained.

2. Evaluating the T cell of the induced pluripotent stem cells and cells differentiated therefrom ２ The value was further compared with the previously measured T ２ The method of claim 1 , further comprising comparing the value to a

3. Passaging induced pluripotent stem cells and cells differentiated therefrom, T ２ The method according to any one of claims 1 to 2, further comprising measuring values ​​to evaluate variations of the passaged induced pluripotent stem cells and cells differentiated therefrom from the induced pluripotent stem cells and cells differentiated therefrom.

4. The method according to any one of claims 1 to 3, wherein the sample is a pellet containing induced pluripotent stem cells and cells differentiated therefrom in the sensor.

5. The method according to any one of claims 1 to 4, wherein the sample comprises a cluster of induced pluripotent stem cells and cells differentiated therefrom.

6. The method of claim 5 , wherein the clusters have a size of at least 50 microns.

7. Said T ２ The method of any one of claims 1 to 6, wherein the value correlates with the doubling time of the induced pluripotent stem cells.

8. Said T ２ The method of any one of claims 1 to 7, wherein the value correlates with the proportion of differentiated progenitor cells.

9. Said T ２ The method of any one of claims 1 to 8, wherein the value correlates with the proportion of differentiated progenitor cells.

10. Said T ２ The method of any one of claims 1 to 9, wherein the value correlates with the proportion of remaining undifferentiated iPSCs in the differentiated cell population.

11. Said T ２ The method of any one of claims 1 to 10, wherein the value correlates with the ability of iPSCs to differentiate into progenitor cells or fully differentiated cells.

12. Said T ２ The method of any one of claims 1 to 11, wherein the value correlates with the expression of OCT4.

13. Said T ２ The method of any one of claims 1 to 12, wherein the value correlates with an EdU colony formation assay.

14. The quality of the induced pluripotent stem cells and the cells differentiated therefrom is determined by the T ２ The method of any one of claims 1 to 13, wherein assessing from the value is performed on day 1 of the differentiation process.

15. A system for evaluating induced pluripotent stem cells and cells differentiated therefrom, comprising: The T of a sample containing a plurality of induced pluripotent stem cells and cells differentiated therefrom is measured in a sensor. ２ and determining the T value of the sample. ２ A system comprising a magnetic resonance relaxometry device configured to evaluate induced pluripotent stem cells and cells differentiated therefrom by monitoring values.

16. The system of claim 15 , further comprising a cell passaging device as a source of the plurality of induced pluripotent stem cells and cells differentiated therefrom.

17. Evaluating the T cell of the induced pluripotent stem cells and cells differentiated therefrom ２ The values ​​were further compared with the previously measured T values ​​of induced pluripotent stem cells and cells differentiated therefrom. ２ The system of claim 15, further comprising comparing the value to the

18. T of passaged induced pluripotent stem cells and cells differentiated therefrom ２ The system according to any one of claims 15 to 17, wherein values ​​are measured to evaluate the variation of the passaged induced pluripotent stem cells from the induced pluripotent stem cells and cells differentiated therefrom.

19. The system according to any one of claims 15 to 18, wherein the sample is a pellet containing induced pluripotent stem cells and cells differentiated therefrom in the sensor.

21. The system according to any one of claims 15 to 19, wherein the sample comprises a cluster of induced pluripotent stem cells and cells differentiated therefrom.

22. 22. The system of claim 21, wherein the clusters have a size of at least 50 microns.

23. Said T ２ The system of any one of claims 15 to 22, wherein the value correlates with the doubling time of the induced pluripotent stem cells.

24. Said T ２ The system of any one of claims 15 to 22, wherein the value correlates to the proportion of differentiated progenitor cells.

25. Said T ２ The system of any one of claims 15 to 24, wherein the value correlates with the proportion of remaining undifferentiated iPSCs in the differentiated cell population.

26. Said T ２ The system of any one of claims 15 to 25, wherein the value correlates with the ability of iPSCs to differentiate into progenitor cells or fully differentiated cells.

27. Said T ２ The system of any one of claims 15 to 26, wherein the value correlates with the expression of OCT4.

28. Said T ２ The system of any one of claims 15 to 26, wherein the value correlates with an EdU colony formation assay.

29. The system according to any one of claims 15 to 28, wherein the induced pluripotent stem cells and cells differentiated therefrom are suitable for application in spinal cord injury cell therapy.

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