Generation of UCiPSCs-Derived Neurospheres for Cell Therapy and its Application in Cell Shipment at Ambient Temperature

Background Neural cells therapy remains one of the most potential approaches for neurological disorders treatment. The discovery of human induced pluripotent stem cells (hiPSCs) and the establishment of hiPSC-derived human neural stem cells (hiNSCs) have revolutionized our technique to cell therapy. Meanwhile, it is often required that NSCs are stored and transported long distances for research or treatment. Although high survival rates could be maintained, conventional methods of cell transport (dry ice or liquid nitrogen) are inconvenient and expensive. Therefore (cid:0) the establishment of a safe, affordable, and frequent obtained hiPSCs and hiNSCs, with characteristics that match fetal hNSCs and a simple, low-cost way to store and transport, are incredibly urgent.


Abstract Background
Neural stem cells NSCs therapy remains one of the most potential approaches for neurological disorders treatment. The discovery of human induced pluripotent stem cells (hiPSCs) and the establishment of hiPSC-derived human neural stem cells (hiNSCs) have revolutionized our technique to cell therapy.
Meanwhile, it is often required that NSCs are stored and transported long distances for research or treatment. Although high survival rates could be maintained, conventional methods of cell transport (dry ice or liquid nitrogen) are inconvenient and expensive. Therefore the establishment of a safe, affordable, and frequent obtained hiPSCs and hiNSCs, with characteristics that match fetal hNSCs and a simple, lowcost way to store and transport, are incredibly urgent.

Methods
We reprogrammed human urinary cells to iPSCs using a virus-free technique and differentiated the iPSCs toward iNSCs/neurospheres and neurons, under Good Manufacturing Practice (GMP)-compatible conditions. The pluripotency of iPSCs and iNSCs was characterized by a series of classical methods (surface markers, karyotype analysis and in vitro and in vivo differentiation capabilities, etc).

Results
Here, our results showed that we successfully generated hiNSCs/neurospheres from more available, noninvasive, and more acceptable urinary cells by a virus-free technique and their differentiation into neural networks. Moreover hiNSCs survived longer as neurospheres at ambient temperature than those cultured in a monolayer. Approximately 7 days, the neural viability remained at > 80%, while hiNSCs cultured in a monolayer died almost immediately. Neurospheres exposed to ambient temperature that were placed under standard culture conditions (37 ℃, 5% CO 2 ) recovered their typical morphology, and retained their ability to proliferate and differentiate.

Conclusions
In this study, we provided a simple method for the storage of NSCs as neurospheres at ambient temperature as an alternative to more costly and inconvenient traditional methods of cryopreservation. This will enable hiNSCs to be transported over long distances at ambient temperature and facilitate the therapeutic application of NSCs as neurospheres without any further treatment.

Background
In regenerative medicine, the clinical use of stem cell products is expected to bring substantial bene ts to patients suffering from various diseases. Limbal stem cells have been registered as a product for eye burns in Europe [1]. Neural Stem Cells (NSCs), multipotent cells that differentiate into the neurons and glia of the central nervous system. Due to their ability to self-renew and differentiate into the nervous tissue, they offer signi cant therapeutic potential in the treatment of neurological diseases such as spinal cord injury (SCI) [2], Alzheimer's disease (AD) [3], and Multiple sclerosis (MS) [4]. However, many currently available cell lines present us with severe obstacles relating to donor tissue acquisition, heterogeneity, availability, and related technical or ethical issues [5]. Besides,it is essential that cells must be transported from one place to another around the world for research and treatment.
So far, many types of human cells have been reprogrammed to iPSCs, including skin broblasts [6], peripheral blood [7], periodontal ligament [8], keratinocytes [9], adipose stem cells [10],etc. Compared to these, Zhou et.al discovered that urinary cells provide us a non-invasive, practical and unlimited source for reprogramming [11]. Since the discovery of induced pluripotent stem cells (iPSCs) [6], induced neural stem cells (iNSCs) have progressed rapidly toward applications in neurodegenerative disease [12]. The availability of patient-speci c iPSCs-derived NSCs may alleviate di culties of immunological issues and ethical concerns. For the systematic clinical use of iNSCs, the wide availability of donor cells would rst be required. Furthermore, the properties of cellular behaviour and the therapeutic e cacy of any iNSC preparations must be reproducible. To achieve this, complete stem cell functional characteristics need to be expressed and stabilized despite the extensive self-renewal of cells.
We have established a method in our laboratory to isolate cells from urine, reprogram them into iPSCs, then induce their differentiation into NSCs. This non-invasive method e ciently produces NSCs for use as a cell culture model to simulate neurological disease. However, human stem cells are susceptible to adverse external conditions, and their transportation relies on expensive and inconvenient cryopreservation. In this study, we showed that iNSCs survive longer as spheroids at ambient temperature than those cultured in a monolayer. we further researched and developed a novel method of NSC preservation that is time-saving and less labor-intensive in comparison with current storage methods, which is conducive to long-distance transportation of cells.

Urinary Cells Collection and Expansion
Zhou et al. detailed the procedure for urinary cells isolation and expansion in 2012 [13]. Collect approximate 200ml urine with sterile 50 ml tubes and centrifuge them at 800g for 5 min at room temperature. Gently and quickly pour out the supernatant, so as not to pour out the precipitate. About 5 ml of each tube is left and mixed into a centrifugal tube. Add 20 ml of washing buffer and centrifuge the samples at 800g for 5 min at room temperature. Carefully remove the supernatant, leaving ~0.2 ml plus the pellet. Add 1 ml of primary culture medium to resuspend the cell pellet, and then transfer the volume into a single well of a 12-well plate (coated beforehand with 0.1% gelatin). Add 1 ml of primary culture medium for the rst 3 days, but do not remove any medium. Approximately 4 days after plating, remove most of the medium, and add 1 ml of REGM medium (LONZA, CC-3190, Switzerland). Next, change half of the medium every day and observe with a microscope until the cell density reaches 80-90%. Split the cells 1:3 or 1:4 to a 6-well plate and UCs should expand quickly.

hiPSCs derivation and maintenance
The iPSCs were acquired by reprogramming the UCs as described in previous work [13]. The iPSCs were reprogrammed from the UCs of a healthy male of 23-year-old using the same method. For the following experiments, all iPSCs were cultured on Matrigel (BD Biosciences, 356234, USA) in mTeSR TM 1 (Stemcell Technologies, 5872, Canada).

Karyotype analysis
Karyotype analysis was performed in iPSCs at passage 15 and in iNSCs at passage 5. When the cells had reached the logarithmic phase, Colcemid was added to a nal concentration of 20 μg/ml for 2 h. The Supernatant was removed, and the pellet resuspended in 8 ml of 0.075 M KCl and incubated for 20 min at 37 °C. The cells were xed with fresh Carnoy's Fixative (3:1 ratio of methanol: glacial acetic acid). Twenty metaphases were analyzed at 450-500 band resolution using Ikaros (MetaStstems, Germany) on an Olympus BX51 microscope.

Teratoma formation
Human iPSC cells were harvested in 1.5 ml tube and two million cells were injected into the ank subcutaneously and Lower limb intramuscularly of NOD/SCID mice. After 8-10 weeks, euthanasia of mice with rising CO 2 levels, tumors were embedded in para n, and sections stained with hematoxilin/eosin and histologically analyzed.

Immuno uorescence staining
Cells were brie y xed in 4% paraformaldehyde for 15 min at room temperature. After permeabilization with 0.5% Triton X-100 in PBS for 5 min, the cells were blocked with 0.5% Triton X-100 and 10% Goat serum for 30 min. Next, the cells were incubated with primary anti-bodiesdiluted in 10% Goat serum at 4°C overnight and then incubated with secondary antibodies (Supplementary Table.2) diluted in 10% Goat serum for 1 h at room temperature. Nuclei were counterstained with DAPI (Beyotime Biotechnology, C1005, China) for 5 min. Images were acquired with an inverted uorescence microscope (Olympus, IX73, Japan). Quantitative Real-time PCR (qRT-PCR) Total RNA was extracted using TRIzol reagent (Invitrogen, 15596026, USA). Total cDNA was prepared with HiScript II Q RT SuperMix for RT-qPCR (Vazyme, R223-01, China). qRT-PCR was then performed using speci c primers in a CFX96 Real-Time System (Bio-Rad, USA). Primers are listed in Supplementary Table  2. Electrophysiology.
Electrophysiological recordings were performed at using a whole-cell, voltage-or current-clamp technique. Whole-cell recordings were made with 6 -9-MΩ borosilicate glass electrodes. Speci c protocols were depicted in each gure. An Axopatch 200B ampli er (Axon Instruments, USA) was used to record the electrophysiological signals. The data were acquired and analyzed using Clamp t 10.

Data analysis
Statistical analyses for all the experimental data was performed using GraphPad Prism 7 and Microsoft Excel. The data were presented as mean ± SD. Statistical signi cance were determined using paired Ttest.

Results
Generation and characterization of UCs-derived hiPSCs Human urinary cells (UCs) were cultured and reprogrammed into induced pluripotent stem cells with a non-integrating, episomal-based system ( Supplementary Fig.1). The iPSCs showed a typical human embryonic stem cell (hESC) morphology and were self-renewable (Fig. 1A) and expressed speci c marker NANOG (Fig. 1B). Quantitative Real-time PCR (qRT-PCR) displayed that the endogenous pluripotency genes (NANOG, OCT4 and SOX2) were at a high expression in hiPSCs and the absence of exogenous expression in parental UCs (Fig. 1C). G-band analysis of the iPSCs (>15 passages) showed normal diploid 46, XY karyotype (Fig. 1D). The NOD-SCID mice were injected with hiPSCs in the underarm skin and hindleg muscles and the capacity for differentiation into three germ layers was certi ed by teratoma formation in vivo, such as cartilage (mesoderm), gut-like epithelium (endoderm) and neural rosette (ectoderm) (Fig. 1E). This hiPSCs were negative for mycoplasma test (Supplementary Table 1). Finally, STR analysis also veri ed the genetic identity of the cell line and the parental urine cells (Supplementary Table. 3). In conclusion we generated a non-invasive, virus-free UCs-derived iPSCs (named C1P4) from human UCs under feeder-free and xeno-free conditions.

Generation and characterization of iPSCs-derived NSCs
We used a highly e cient method to induce NSCs (iNSCs) from iPSCs and their differentiation into neurons. The steps of the large-scale generation of iNSCs and neurons using embryoid bodies methods (EBs) are schematically described in Fig. 2A. iPSCs are ampli ed for 4 days in mTeSR TM 1 medium.
Undifferentiated hiPSC colonies were broke into fragments using a P1000 pipette and re-plated onto Matrigel-free dishes in EBs medium to generate EBs for 4 days. The EBs were re-plated onto Matrigelcoated dishes. Neural rosettes were visible and matured within 14 days. Rosettes were picked and then dissociated into single cells with Accutase that are suspended in culture. After 7-10 days, the single cells produced round neurospheres (Fig. 2B). In order to determine the actual fate commitment of the generated neurospheres/iNSCs, we performed further differentiation into neurons (Fig. 3A).
To prove the presence of iNSCs, we performed immunocytochemical staining for NSC markers (PAX6, Nestin). The iNSCs showed positive PAX6, Nestin staining (Fig. 2C). G-band analysis of the iNSCs (>5 passages) showed normal diploid 46, XY karyotype (Fig. 2D). Next, to exclude the permanence of cells expressing hiPSC markers in iNSC cultures, the expression of iPSC markers in iNSCs was compared to parental iPSCs. Unlike iNSCs, iPSCs expressed detectable levels of pluripotency markers (OCT4, NANOG and SOX2) (Fig. 3E). In contrast, NSC-positive marker Nestin and PAX6 were expressed, but neuronpositive marker Map2 was not expressed by iNSCs (Fig. 3F). To preclude the presence of nonneuroectoderm cells, we compared the expression of mesodermal, endodermal and neuroectodermal cell line markers of iNSC to EBs. iNSCs did not express GATA4, AFP (entoderm) and TBX1 (mesoderm), but maintained a high expression of PAX6 and SOX1 (ectoderm) (Fig. 3G). Therefore, we obtained a cell line expressing only the properties of iNSCs by the EBs method.

iNSCs differentiate into cerebral cortical neurons
In order to con rm that the iNSCs has the characteristics of functional progenitors, we differentiated the iNSCs into mature cerebral cortical neurons and neural networks. We used a method in the previous protocol [14] and single-celled iNSCs at passage 5 were plated onto Matrigel-coated dishes in neural differentiation medium for 30 days before performing immuno uorescent staining. To identify the mature neurons obtained, we used neuron-speci c cytoskeletal marker TUJ1, dendritic marker Map2 and astrocyte marker GFAP. The iNSCs successfully differentiated into cortical neuron and astrocyte, as con rmed by the presence of staining for the cortical neuron and astrocyte markers (Fig. 3B-D). Moreover, whole-cell patch-clamp recordings were performed to assay the functional maturity of the iNSCs-derived neuronal networks (Fig. 3E-G).

Neurospheres formation extends iNSCs survival under ambient temperature
In this study we aimed to understand the effect of ambient temperature (AT) treatment on the viability of iNSCs and establish a simple approach for cell storage and shipment. Human iPSCs were induced to differentiate into neurospheres using the method we mentioned previously (Fig. 2). Neurospheres were then collected, put into a 15ml centrifuge tube, and placed on the cell room under AT for 7 days. To prove that the neurospheres still has the characteristics of functional progenitors after 7 days, we rst performed the qRT-PCR and the result showed the neurospheres/Day7 still express positive markers (PAX6, Nestin) of NSCs ( Supplementary Fig.2). Meanwhile, we differentiated the neurospheres into mature cerebral cortical neurons. Immuno uorescence results showed that the neurospheres still differentiate into neurons (Fig. 4A). In contrast, iNSCs cultured in monolayer in a 6-well plate under AT for 7 days as well. monolayer-iNSCs quickly oated up and then died within three days. Conversely, neurospheres did not change signi cantly in morphology within 7 days under AT. The survival rate dropped rapidly on day 9 (Fig. 4B). In order to test whether neurospheres can withstand temperatures other than ambient temperature, neurospheres was respectively stored at 10, 20 and 30℃, and the survival rate of neurospheres basically unchanged (Fig. 4C). The survival rates of neurospheres/AT and monolayer-iNSCs/AT were compared with those of monolayer-iNSCs thawed after conventional cryopreservation. The result revealed monolayer-iNSCs cannot tolerate ambient temperatures for long periods of time. On the other hand, neurospheres/AT and cryopreservation achieved similar survival rates ( Figure 4D).

Discussion And Conclusions
Nervous system diseases, such as spinal cord injury (SCI), autism spectrum disorder (ASD) are associated with the dysfunctional recovery and limited regenerative capacity of the central nervous system (CNS), which cannot repair or replace neurons and axons after injury. Transplantation of stem cells, especially neural stem cells (NSC), can repair or replace damaged neurons and glial cells by providing a suitable microenvironment and enhancing their regeneration. Substantial preclinical researches have furthered the use of Neural Precursor Cells in clinical trials [15][16][17]. A preclinical study showed that NSCs induced by iPSCs in healthy 86-year-old males still function in transplantation therapy [18].
iPSCs produced by reprogramming adult cells into a self-renewing pluripotent state eliminate the ethical issues associated with the use of human fetal/embryonic tissue and reduce immune rejection of implanted cells. UCs are exfoliated epithelial cells of the kidney system and can be collected in any condition except renal failure [13]. Thus, urine shed cells provide us with a practical and unlimited source of human cells for reprogramming, and this non-invasive approach to human cell acquisition will signi cantly improve patient compliance. Besides, epithelial to mesenchymal transformation (EMT) is essential for somatic cells to become stem cells, and UC, as a renal epithelial cell, is easier to overcome this transformation due to its epithelial origin [11]. Therefore, UCs are a very useful tool for studying cell therapy and tissue engineering.
Although numerous studies have demonstrated the clinical potential of iPSCs-derived NSCs in treating neurological diseases, there are still some problems and challenges. The iPSC/iNSC generation method needs to be better optimized to produce cells with good therapeutic e cacy and minimal adverse side effects, so as to make them more valuable for clinical use. The clinical use of iNSCs depends mainly on the availability of a suitable donor cell source, which must remain reproducible and predictable enough to produce the desired number of cells. This large-scale production ensures uncompromised cellular therapy of patients. Furthermore, temperature is also one of the main in uencing factors for the survival of cells in vitro and high viability after thawing is an urgent requirement for cell transportation [19].
Combination of Dorsomorphin (BMP signals inhibitor) and SB431542 (TGFb/activin/nodal signals inhibitor) promoted neural induction [20]. In this study, by using this combination in the rst step of the differentiation scheme, we discovered the formation of representative rosette structures of early-stage ESor iPSC-derived neural precursors, and then generated and characterized a population of virus-free hiPSCs-derived hiNSCs expressing typical NSC markers (PAX6 and Nestin) and further differentiated them into neurons that exhibit a characteristic feature. Like ES-derived iNSCs, our iNSCs had a normal karyotype and settled ampli cation e ciency, did not possess telomerase activity. Our iNSCs preparation demands one month, followed by another one month for expansion. Thus, our protocol can produce a large amount of stably iNSCs for several serial transplants from clinical-grade UCs-derived iPSCs.
Cells need to be frozen and transported for any particular use. For long time storage, the traditional cell cryopreservation method requires -80℃ refrigerator or liquid nitrogen long time. Moreover, conventional transportation methods require a particular container, including dry ice or liquid nitrogen, to keep low temperatures. Neural stem cells also need to be preserved like this. But with traditional preservation, it's very di cult to ensure cells survived at room temperature for a long time. Bin Jiang et.al found spheroidal formation preserves MSCs for prolonged time under ambient conditions for facile storage and transportation [21]. We demonstrated the feasibility of iNSCs as neurospheres storage and transportation at ambient temperature. This will remarkably facilitate long-distance transportation and therapeutic application of iNSCs without further processing. In our experiments, we found that neurosphere could be stored for about 7 days, and the survival rate could reach more than 80%. This method of transporting neural stem cells has the advantages of being simple, low cost, and does not require special containers or dry ice. The living cells can be quickly recovered and put into working state after being transported to the destination. Cell viability is also acceptable compared to traditional methods. Therefore, this neurosphere preservation method meets the cell transportation between cities and countries as well.
In conclusion, generating iNSCs from UCs-derived iPSCs and the formation of the neurosphere for transportation and storage as described here may have several distinct advantages. First, urine collection is simple and straight-forward compared with other sources of human biological material, which simpli es ethical issues. Second, the application of NSCs was commonly under 10 passages for maintaining relatively high stemness [22]. UCs-derived iPSCs could guarantee that the number of iNSCs is almost unlimited for treatment of neurological diseases. Third, the form of neurosphere is conducive to transportation and storage at ambient temperature, and it is easy to scale production. Fig.5 summarizes the whole process schematically from reprogramming to the application of neurospheres. However, there are still some problems. We still need a lot of animal experiments to prove its feasibility in vivo before it can be used in clinical stage. The data that support the ndings of this study are available from the corresponding author upon reasonable request.

Contributions
LS and participated in the study design, performed the main laboratory work and statistical analysis, prepared the drafts of the manuscript, and revised the manuscript according to advice from the other authors. LZY participated in the study design and revised the manuscript. ZHF participated in the laboratory work, performed the statistical analysis. HXB, HL, LZX, HRQ, TC, TF, HHL and OM participated in the study design and helped to revise the manuscript. All authors read and approved the nal manuscript.

Corresponding author
Correspondence to Zhiyuan Li.

Ethics approval and consent to participate
The individual has signed written informed consent for donating UCs in this study. The experiments involving human subject and animal research had been reviewed and approved by the Human or Animal Subjects Research Ethics Committee/IRB, Guangzhou Institutes of Biomedicine and Health (GIBH), Chinese Academy of Sciences.

Consent for publication
Not applicable.

Competing interests
The authors declare no con ict of interest.    Neurospheres strengthen survival of AT-exposed iNSCs. A.iNSCs-monolayer were cultured to form neurospheres and exposed to AT for D3 and D7. The neurospheres/D7 were isolated and then