Ultrasensitive in Vivo Fluorescence Imaging of Mesenchymal Stem Cells and Small Molecule-Induced Pluripotent Stem Cells in BALB/c mice.

Background Mesenchymal cell has been frequently used in clinical studies. Mesenchymal stem cells (MSCs) are self-renewing, multipotent stem cells with the potential to differentiate into multiple mesoderm lineages. But MSC have limitation in clinical application for treating human diseases because they can differentiate several types of cell but not all types. PSL (Pluripotent Stem cell Like cell) are newly developed pluripotent stem cells from human mesenchymal stem cells (hMSC) induced by small molecule compounds. These cells have potential advantages for clinical cell treatment compared with ESCs and iPSCs. Methods We induced pluripotency from MSC using small molecules. It has tried to trace MSC and PSL in mice using bioluminescent techniques, which can detect visible light emitted from cells labeled with miRNA conjugated uorescent molecules.


Abstract
Background Mesenchymal cell has been frequently used in clinical studies. Mesenchymal stem cells (MSCs) are selfrenewing, multipotent stem cells with the potential to differentiate into multiple mesoderm lineages. But MSC have limitation in clinical application for treating human diseases because they can differentiate several types of cell but not all types. PSL (Pluripotent Stem cell Like cell) are newly developed pluripotent stem cells from human mesenchymal stem cells (hMSC) induced by small molecule compounds. These cells have potential advantages for clinical cell treatment compared with ESCs and iPSCs.

Methods
We induced pluripotency from MSC using small molecules. It has tried to trace MSC and PSL in mice using bioluminescent techniques, which can detect visible light emitted from cells labeled with miRNA conjugated uorescent molecules.

Results
MSCs predominantly migrate into the brain and testis. They also migrate to the liver, omentum, mesentery, kidneys and spleen. Migration of PSL is similar to MSCs, in that they go to the brain, testis and other intraperitoneal organs. Fluorescent images of explanted organs show that the intensity of brain images is higher in the PSL mouse group than the MSC mouse group. However, testis, image intensity is higher in MSC mouse group than the PSL mouse group. In PSL but not MSC mice, uorescence persisted at the injection site in the tail.

Conclusions
In this study, injected MSCs and PSL predominantly migrated to the brain and testis. But, PSL migration was more than MSC migration in Brain. Both cell types had a similar migration pattern except for persistent uorescence at injection site in the tail vein of PSL mice. We expect these cell therapy may have many potentials for clinical studies on these notable treatments.

Background
Stem cell-based therapies are being actively explored as a potentially innovative therapeutic strategy for various genetic and acquired diseases.
Recently, the reprogramming of human somatic cells into human-induced pluripotent stem cells (hiPSCs) has been reported. As hiPSCs can differentiate into all cell lineages this has opened new opportunities for damaged organ repair 1,2 . Although many hiPSCs clinical studies have been carried out on various disease, there are only a few reports that it has been effective at curing disease. One problem is that Page 3/15 hiPSCs are made with genetic mutations which may have tumorigenic potential 3 . Another problem is the low yield of hiPSCs, as it is important to administer enough stem cells to be effective.
We have recently developed pluripotent stem cells from umbilical cord, adipose tissue, cord blood and bone marrow MSCs. We named these pluripotent stem cells as PSL (U.S. Patent No. 10,131,881., 2018).
PSL (Pluripotent Stem cell Like cell) are pluripotent stem cells induced from human mesenchymal stem cells (hMSC) by the addition of the small molecule compounds. This is a novel approach for inducing pluripotency. PSL are pluripotent stem cells like as ES and iPSCs. The advantage of PSL compared to other hiPSCs is that these cells have no mutational potential and can be produced with a high yield rate.
Recently, there has been a report on chemically induced pluripotent stem cells (CiPSCs) which also can be induced by small molecule compounds 4 . The CiPSCs in this report originate from mice, while the PSL in this study are the rst chemically induced human pluripotent stem cells developed from human cells. Mesenchymal cell has been frequently used in clinical studies. Mesenchymal stem cells (MSCs) are selfrenewing, multipotent stem cells with the potential to differentiate into multiple mesoderm lineages 5,6,7,8 .
MSCs are non-immunogenic 9 and regarded as a promising source for cell-based treatments of various complicated disease, such as cardio cerebrovascular disease 10,11 , neuronal injury 12,13 , hepatic diseases 14 and respiratory disease 15 . And another character of MSCs is their immunosuppressive effect in graftversus-host disease 16,17 . The pathological mechanisms of MSCs have been well studied in vitro. However, in vivo studies are still rare and the fate and survival of MSCs in vivo are not well understood. In contrast, the use of iPSCs has rarely been reported in clinical trials. To know the exact physiological mechanisms of MSCs or PSL, tracking and localization of these cells in vivo is necessary. If these cells can be tracked, the healing and reconstructive process can be studied.
Fluorescence Imaging (FI) methods can detect visible light emitted from cells labeled with miRNA conjugated uorescence molecules 18,19,20 . FI can track cells implanted in living animal non-invasively and in real time 21 . This technology has been used in this study to trace and localize MSCs and PSL.
Using this technique, gross photos were obtained to localize the accumulation of these cells in vivo. The animals were sacri ced and specimens extracted to provide localized uorescent images from organs including the brain, liver, heart, spleen, kidney, testis, lung, adipose tissue, omentum and mesentery.
There have been a few reports on MSC localization and tracing after administration 22,23 . This study on the localization of PSL and MSCs may be useful for future applications in clinical trials.

Preparation of MSCs
Human umbilical cords were obtained from full-term Caesarean section deliveries and then stored in Dulbecco's modi ed Eagle medium (DMEM)/F12 (1:1) supplemented with 100 U/ml penicillin and 100 μg/ml streptomycin (GIBCO, Invitrogen Inc. Carlsbad, CA, USA). To isolate stem cells, the cord was cut into 4-5 cm long pieces, and the vessels were pulled away to leave only Wharton's Jelly (WJ). WJ was cut into 1-2-mm3 pieces and digested with 1 mg/ml of collagenase type 1 (Millipore Sigma, St. Louis, MO, USA) in phosphate-buffered saline (PBS) at 37 °C for 30 min. The digested mixture was then passed through a 100-μm lter (BD Biosciences, Franklin Lakes, NJ, USA) to obtain cell suspensions. The cells were washed with PBS solution and then cultured in DMEM/F12 medium containing 10 % fetal bovine serum, 2 mmol/L glutamine, 1 % nonessential amino acids, and 1 % penicillin/streptomycin (GIBCO, Invitrogen Inc., Carlsbad, CA, USA) at 37 °C and 5 % CO 2 . Non adherent cells were removed when the medium was changed on day 3. The multipotent MSCs, which were adhering to the plastic bottom were then collected. The multipotent MSCs phenotype is de ned as the co-expression of antigens (CD105, CD73, and CD90 [≥95 % positive]) and the absence of hematopoietic lineage markers (CD45 and CD34 [≤2 % positive]). In summary, multipotent MSCs phenotype was con rmed by positive expression markers (CD105 CD73 and CD90-FITC) and negative expression markers (CD45 and CD34-FITC).

Preparation of PSL Cells from MSCs
We used small molecular compounds (STC-F002), extracted from sea algae (Eklonia cava). The MSCs was cultured in DMEM-F12 medium containing 10% FBS. When 90% of the dish bottom was covered with these cells, approximately day 5, 10ug/mL of STC-F002 was added to the culture medium (U.S. Patent No. 10,131,881(2018)).
Approximately 10 days later, distinct types of colonies appeared which looked similar to human Embryonic Stem cell (hES) colonies. The hES cell like colonies were carefully picked and mechanically disaggregated into small clumps.

Expression of Pluripotent Stem Cell Markers in PSL
The PSL cells strongly expressed all the characteristic pluripotent stem cell markers. These included SSEA4, a cell-surface glycosphingolipid used for detecting human ES cells; Oct3/4, a protein involved in the self-renewal of human ES cells, Sox2, a transcription factor that controls genes involved in embryonic development and AP, ubiquitous membrane-bound glycoprotein for detecting pluripotent stem cell 40 . In comparison MSCs were either negative or weakly positive for the pluripotent stem cell markers.
Step 2 medium consisted of DMEM-F12 supplemented with 2% FBS and 25ng/ml BDNF. Neurogenesis was assessed on day 6 and day 10, and imaging for nestin was performed on day 12. Synthesized miR-221 MB probe was con rmed with selectivity assay by uorescent spectrometry 37 .
The miR-221 MB probe was incubated with the MSCs and PSL cells check the expression of intracellular miR-221 in a tube for 5 min at room temperature. miR-221 MB was also able to bind to miR-221 in the human MSCs and PSL cells from different pathological origins.

In vivo imaging
MSCs and PSL cells were traced in vivo with the miRNA conjugated uorescent dye. The miRNA conjugated uorescent dye system was conducted and transferred to MSCs and PSL cells. Mice were anesthetized before MSCs and PSL cells were injected. Cell transplantation was conducted as described above. miRNA-221 was tracked from approximately 30 min after miRNA221-labeled MSCs and PSL cell injection. Near-infrared (NIR) uorescence images (595/625 nm) of the mice were taken using an IVIS Spectrum 2D in vivo imaging system (Perkin Elmer, Connecticut, USA) to monitor miRNA-221 37 . cell tracing was performed at about 10 min after substrate injection.
Imaging analysis were normalized mice imaging with MB probe injection to eliminate signal interference using mice imaging without cell injection Immunohistochemical analysis of animal tissues Samples from each organ were snap-frozen in OCT compounds (Sakura). Cryosections (5-15 μm) were air-dried and then xed in 4% paraformaldehyde (Seongnam, Bioseasang, Korea) for 10 min.
After blocking with 5% (v/v) goat serum (Life Technologies) for 1 h at room temperature, the samples were incubated with the primary SSEA4 antibody (Santacruz, USA) for 12h at 4 °C. After washing in PBS, they were stained with the secondary antibody and H33342 (Life Technology, Eugene, USA) for 30 min at room temperature. Immune staining was observed and recorded using a Zeiss LSM 800 confocal laser microscope with a 20x NA 0.6 objective (Carl Zeiss Microscopy GmbH). The Emission range of the lasers was 405 nm for H33342 and 625 nm for miRNA-221-MB. The microscopy image data was reconstructed using a maximum intensity projection algorithm implemented in the Zeiss ZEN lite blue 2.5 software (Carl Zeiss Microscopy GmbH).

Characterization of MSCs
MSCs cells were highly positive for CD105, CD73, and CD90 but negative for CD45 and CD34 (Figures 1A) 24

PSL induction by small molecules
MSCs cultured with small molecules (STC-F002) for 7-10 days formed compact colonies that were positive for Oct3/4, SOX2 SSEA4 and AP (Alkaline phosphatase) expression (Figures 2A, C). Both the PSL colonies and individual PSL cells strongly expressed all of the characteristic pluripotent stem cell markers that were examined. These included Oct3/4, Sox2, SSEA4 and AP. MSCs derived from Wharton's jelly tissue were either negative or weakly positive for these pluripotent stem cell markers To complete our examination of the potential for spontaneous differentiation into the three germ lines, PSL cells were differentiated into mesodermal lineages using chondgenic and osteogenic differentiation medium. Formation of chondrocytes was detected using alcialn blue and formation of ostreoblasts was detected using Von Kossa stain. (Figures 2B) 25 . PSL have also shown the ability to differentiate into neuronal cells lineages. Neural-like cells were detected by immuno uorescence using anti-human nestin antibodies. PSL cells showed signi cant expression of nestin as shown by H33342 staining (Figures 2B), con rming their potential to spontaneously differentiate into ectodermal cells, unlike the control MSCs which were negative for nestin. PSL cells were differentiated into endodermal cells by exposing them to hepatocyte differentiation medium for 7 days. The differentiation of PSL cells were con rmed by alphafetoprotein, which is expressed during the development of the endoderm and by the progenitors of hepatocytes( Figures 2B). These results demonstrate that PSL are pluripotent stem cells capable of differentiation to induced ectodermal, mesodermal and endodermal cells in culture.

Gross uorescent in vivo images of MSCs and PSL
Early uorescent images of both groups show the accumulation of cells around the pleural and lung area of the prone photo. They gradually spread to other areas and become fainter over time (Figures 3A, D). Fluorescence images show accumulation into the peritoneal area and which persists until the animals were sacri ced in the supine photo. The peritoneal area may include the liver, omentum, mesentery, kidneys and spleen.
Fluorescence was also seen in brain area in both groups. The intensity of uorescence in the brain was higher in the PSL group. A uorescence signal at injection site in the tail persisted for the entire experiment (9 day) in PSL mice. In contrast, no uorescent signal at the injection site was observed in MSC mice ( Figures 3A, D arrow).

Fluorescent images of an explanted organ with MSCs and PSL
When uorescence intensity was measured in explanted organs, intensity was brighter in the brain and testis of both MSCs and PSL administered mice. But uorescence intensity of PSL was higher than uorescence intensity of MSC. In MSC mice, increased uorescence intensity was also found in the liver, omentum, mesentery, kidneys and spleen. In PSL mice, increased uorescence was also found in the liver, omentum, mesentery, spleen, kidney, lung and adipose tissue around the testis. Fluorescence was barely present in lung and adipose tissue of MSC mice ( Figures 3B, C, E, F, G).
Histological identi cation of transplanted MSCs and PSL SSEA4 staining was done on brain and testis tissue to identify MSCs and PSL. SSEA4 staining was positive only in the brains and testis of mice administered with PSL. In contrast, SSEA4 staining was negative in MSC mice. As SSEA4 is the marker for human pluripotent stem cells, SSEA4 staining in the brain and testis means that the administered PSLs, which are human pluripotent stem cells, have accumulated in those organs.
Brain and testis tissues of both MSCs and PSL mice were miRNA221-cy5.5 positive. The intensity of miRNA221-cy5.5 in brain tissue was stronger in PSL mice. However, its intensity in testis tissue, was higher in MSC mice (Figures 4A, B).

Discussion
There have been many reports on the e cacy of MSCs and PSCs in treating various diseases 26,27,28,29,1 . Previously, it was believed that PSCs repair damaged tissues through differentiation in injured organs. Therefore, to deliver greater numbers of PSCs to injured tissues, researchers have attempted diverse injection strategies. The best way to deliver PSCs is intravenous administration. After administration, the PSCs are distributed throughout the body and signi cant portion presumably reach the damaged organ.
However, there have been few studies tracing and localizing PSCs 30,31 . There has been some reports on the tracing of MSCs in mice. In this study, we tried to trace MSCs and PSL in mice using bioluminescent techniques. The MSCs used were obtained from umbilical cords. These MSCs were identi ed as positive for CD73, CD90 and CD105 and negative for CD34 and CD45. They also differentiated into adipose cells and chondrocytes. PSLs were induced from MSCs with small molecule compounds (STC-F002) and identi ed with the human pluripotent markers SSEA4, OCT4 and SOX2. PSL was differentiated into three germ layer cells. Differentiation into ectodermal (neuronal cell), endodermal (hepatocyte) and mesodermal cells (osteoblast and chondrocyte) were demonstrated in this study. PSL has the advantages over other PSCs such as ESCs and iPSCs. The most important problem with ESCs is an ethical concern that the derivation of pluripotent stem cell lines from oocytes and embryos is fraught with disputes. Because of this problem ESCs are rarely used in clinical trials. Another problem is the possibility of malignant transformation. iPSCs also have a problem with becoming cancerous. As iPSCs pluripotency is induced using gene insertion, they also have the potential for malignant transformation. However, the pluripotency of PSL are induced by small molecules without gene insertion and so have no mutational potential. Another advantage of PSL is that massive production is possible due to their high yield rate. CiPSCs are similar to PSL in that their pluripotency is induced by small molecules. However, CiPSCs are still of mouse origin and have a very low production yield rate.
The PSL used in this study are the rst PSCs of human origin induced by small molecule compounds. There are reports on administering MSCs to mice. It was been shown there is no interaction between MSC and the immune system of mice 32 . Similarly, human PSL are also expected to have no immunology reaction with mice. When MSCs and PSL were administered to mice, there was no immunologic interaction in this study.
The migration of injected MSCs and PSCs remains unknown. If the migration, localization and fate of this cells are known, the physiologic process of healing or repairing can be studied. There has also been one report on the migration of MSCs in mice. They have shown that MSCs migrate to the lungs, kidneys, and skin of the lower back. In our study, MSCs predominantly migrated to the brain and testis though they also migrated to the liver, omentum, mesentery, kidneys and spleen. Migration of PSL was similar pattern with MSCs in that they went to the brain, testis and other intraperitoneal organs, but PSL migration was more than MSC migration in Brain. This is the rst study to show that these cells migrate to the brain and testis. Fluorescent images of explanted organs had higher intensity in the PSL mouse group than the MSC mouse group in the brain. However, in the testis, intensity was higher in the MSC mouse group than the PSL mouse group.
This migration to the brain and testis by PSL was also con rmed by staining for SSEA4, which is a marker for human pluripotent stem cells.
It is unclear why MSCs and PSL predominantly migrate to the brain. There must be some still unknown interaction between these cells and the brain. The amount of stem cells present in the body decreases with age, which may be related to a decrease in organ function 33 . One of the organs susceptible to decreased stem cells might be the brain which may result in dementia 34 . The interaction of PSCs and the brain may be vital for proper brain function.
It is also interesting that MSCs and PSL migrate to the testis. The testis is the locus of reproduction in male animals. The accumulation of these cells in the testis may have some function in reproduction. Reproductive function also decreases with age which is associated with decreased stem cell production.
It is still unknown why stem cell migrates actively into the brain. We presume that brain plays important role in activity of stem cell, although speci c activity is should be studied further. The brain uses 25% of oxygen consumed in whole body. And brain uses 50% glucose consumed in whole body. Accordingly, brain may need substantial an amount of regenerative cells like stem cell. After stem cells enter into brain, they may be involved in the function and maintenance of the brain. We do not know why stem cell enters into testis massively either. The testis is organ where cell division is occurring abundantly. Normal cells have limitation of permanent cell division which is not suitable for reproduction. Stem cells have no limitation of cell division which is necessary for characteristic reproductive organ. Reproductive organ is vital for maintenance of the genes for future generation.
These cells also migrate to the liver, kidney, and spleen, which are important organs in homeostasis.
These organs are also supplied with large amount of blood which may be related with their active metabolic processes. The accumulation of the cells in these organs is related to metabolism and their own function. Although previous reports on MSCs migration has shown that MSCs accumulate in the lung, there was no accumulation of these cells in the explanted lung in our study. One possible reason is that explanted lung was procured after sacri cing the animals on day 9. The cells present in the lung may have escaped the lung during the later days of the experiment. In a prone in vivo image uorescence was shown around the lung during the initial stage.
It is also interesting these cells accumulated in the omentum and mesentery which invites further study.
In PSL but not MSCs mice, uorescence was persistent at the injection site in the tail. It is also unknown why this discrepancy occurs. The injection site can be regarded as an injury area. Therefore, persistent uorescence at the injection site may show the attraction of PSL to the injury site. However, further study of this phenomenon is necessary.
miRNA-uorescence was used to trace the MSCs and PSL. This technique provides ultra-selective binding to target DNA which can be tracked by optical monitoring of visual imaging. Fluorescence imaging method in this study is very sensitive to detect pico mole of target miRNA with high speci city 35,36,37 . Synthesized molecular beacon conjugated with uorescent was made for complimentary reaction with miRNA-221. This imaging technique can detect cell localization in in vivo 37 .

Conclusion
In this study, we found that MSCs and PSL predominantly migrate to the brain and testis. These cells also migrate to the liver, kidney, spleen, mesentery and omentum. The interactions of these cell with each organ are not yet known and further study is necessary to determine their roles. MSC and PSL treatment could be a breakthrough for many incurable diseases. We expect there may be many future studies on these notable treatments.

Declarations
Availability of data and materials The datasets used and /or analyzed during the current study are available form the corresponding author on reasonable request.