Effects Of Intracellular Magnetic Labeling Of Stem Cells With Superparamagnetic Iron Oxide On The Differentiation Capability Into Hepatocyte-like Cells

BackgroundMany acute or chronic end-stage liver diseases have a very high mortality rate, which are intractable. Hepatocyte transplantation as an alternative to whole liver transplantation for the treatment of hepatic diseases is still hampered by the limited availability of marginal donor organs to isolate human adult hepatocytes in sufficient amounts and quality for transplantation. The ready availability and unrestricted potential to propagate and differentiate make stem cells of extrahepatic origin a feasible option to generate hepatocytes for therapeutic application in liver diseases. It has been shown in a number of recent studies that murine and human stem cells from various sources may develop into hepatocyte-like cells in vitro and in vivo. Candidates for such strategies include embryonic stem cells (ESCs), bone marrow mesenchyme stem cells (BMSCs) and adipose tissue-derived stem cells(ADSCs), and so on. Although the therapeutic potential of ESCs is enormous due to their auto-reproducibility and pluripotentiality, there are still some limitations to their practical use, including cell regulations, ethical considerations and genetic manipulation. BMSCs possess adipogenic, osteogenic, chondrogenic, myogenic and neurogenic potential in vitro. Since Zuk et al reported that the stem cells in the body fat tissue was discovered at first time in 2001, the ADSCs became an important source of stem cells gradually. ADSCs with similar characteristics to BMSCs, by nature, immunocompatible, and there are no ethical issues related to their use. Adipose tissue drepresents an abundant and accessible source of adult stem cells that can differentiate along multiple lineage pathways, including adipogenic, osteogenic, chondrogenic, and hepatocyte-like cells in a lineage-specific culture medium. It broughts the hope to the patients who suffered form liver diseases of final stage.The evaluation of stem cell therapy needs to reflect the situation of differentiation, distribution, metastasization and homing of transplanted stem cells in vivo. A number of recent studies show that MRI is the best imaging technique for tracking grafted stem cell in vivo. However, the cells have to be labeled magnetically prior to transplantation to be visible on MRI. At present, superparamagnetic iron oxide (SPIO) is one of the paramagnetic materials which commonly used. However, unmodified form of SPIO particles can not be used to efficiently label stem cells. Therefore, various approaches to label stems cell have been extensively explored. Substantial development has been made by introduction of commercially available transfection agents (TAs), including lipofectamine, poly-L-lysine protamine sulfate, arginine/lysine-rich cationic peptides, dendrimers and silica and alkoxysilane coating, allowing for nonspecific magnetic intracellular labeling with SPIO agents. And poly-1-lysine (PLL) is used mostly at present. Due to their strong harassing effect on local magnetic field homogeneity, iron-based contrast agents as SPIO can be readily detected by MRI, it shows hypo-intensity on T2WI or T2*WI.So it can distinguish grafted stem cells from surrounding tissue easily, and judge the state of labled cells according to the change of MR signal change on target organ. However, the effects of this label technique on viability and differentiation of stem cells still exist disputes. Herein, we have made ADSCs an investigation object, by inducing ADSCs differentiation into hepatocyte-like cells, evaluating cell viability at the same time, detecing glucogen depositions and expression of ALB after differentiation, in order to investigate the effects of intracellular magnetic labeling of ADSCs with SPIO on the differentiation capability into hepatocyte-like cells, and to provide experiment evidence for SPIO application in the field of stem cells transplanted therapy.PurposeTo investigate:(1) separate, cultivate and identify the primitive ADSCs from SD rat; (2) the feasibility of intracellular magnetic labeling of stem cells with SPIO using transfection agent PLL, and to analyze the effect of the viability of the labeling stem cells.; and (3) the effect of this approach on the differentiation capability into hepatocyte-like cells of the SPIO labeled stem cells.Materials and Methods1. Culture and identify the rat's ADSCs in vitro. The fat was obtained from the inguinal area of Sague-Dawley rats, then the primary stem cells were pick-up, and cultured, and identified, and subcultured. The cell surface markers such as CD29, CD45, CD44, CD34 and CD31 of rat's ADSCs were identified with Flow Cytometer.2. To label the stem cells with SPIO and transfection agent PLL, and to analyze the effect of the viability of the labeled stem cells. The passage 3 cells of the rat ADSCs were used. The approved, commercially available SPIO, ferucarbotran (Fe 28mg/ml,Resovist; Schering, Berlin, Germany), was used as the intracellular magnetic labeling agentr. The cells were mixtured with the SPIO- PLL complex (SPIO 25μg/mL and PLL 0.75μg/mL) in the serum free media, and shake up about 30min at 30r/min in the room temperature. The cultures with the cells adherenced to bottol bottom in 90% were kept overnight at 37℃in a couveuse contained 5% CO2. Prussian blue staining was used to evaluate glycogen and intracellular iron uptake. After incubation, the ADSCs were washed with phosphate-buffered saline three times to remove excess SPIO-PLL. Cell viability of the unlabeled and labeled ADSCs was assessed with trypan blue testing.3. The effect of intracellular magnetic labeling of stem cells with SPIO on the cell differentiation capability into hepatocyte-like cells was evaluated. We use the method in part two to label the rat ADSCs with SPIO, and trypan blue testing to evaluate cell viability of the unlabeled and labeled ADSCs. SPIO-labeled and unlabled ADSCs were subjected to differentiate into hepatocyte-like cells. And the protocol as following:the stem cells (passage 4) were divided into four groups. Group 1 was labeled-induced ADSCs, group 2 was unlabeled-induced ADSCs, group 3 was labeled-uninduced ADSCs, and group 4 was unlabeld-uninduced ADSCs. The ADSCs of group 1 and 2 were subjected to hepatocyte-like cell induction. When ADSCs cultured to Passage 4 adhered each other at 80-90%, these ADSCs were used for differentiation assays. Cells were serum deprived for 2d and pre-cultured in LG-DMEM supplemented with 20 ng/mL EGF and 10 ng/mLβ-FGF (conditioning step) to stop cell proliferation, prior to induction of differentiation toward a hepatic phenotype. Then a 2-step differentiation protocol was performed, followed by a sequential addition of growth factors, cytokines and hormones. In step-1 differentiation medium, consisting of LG-DMEM supplemented with 20 ng/mL HGF,10 ng/mLβ-FGF, the cells were cultured for 14d, followed by step-2 differentiation medium, consisting of LG-DMEM supplemented with 20 ng/mL HGF,1μmol/L dexamethasone to achieve cell maturation up to 21d. For each step, the culture medium was replaced every 3 days. Then Periodic acid-schiff (PAS) staining was used to test the glycogen storage within cytoplasm, immunohisto chemistry staining was used to detect the ALB and RT-PCR to identify the specific gene ALB of hepatocyte cells. After inducing 21 days, PAS staining together with Prussian blue staining was used to evaluate glycogen and intracellular iron.Results1. The rat's ADSCs were separated and cultured successfully in vitro. The cell surface phenotyping of ADSCs were identified by Flow Cytometer, which expressed CD 44(100%), CD 29 (99.9%), CD45(1.4%), CD34(7.8%) and CD 31(13.1%).2. It is easy and high-performance for intracellular magnetic labeling of ADSCs with PLL mediating SPIO particles. After PLL-SPIO labeling, instant Prussian blue dye staining of the Labeled ADSCs revealed that most ADSCs (in 100%) were covered with blue iron particles at the end of the labeling process. Trypan blue exclusion testing revealed a mean viability of 92.98±0.588% for the control cells, and 93.50±0.469% for unlabeled cells. No significant difference of the cell viability between the two groups (t=1.683, P=0.123) was found.3. Both of the labeled-induced group and unlabeled-induced group were differentiated into hepatocyte-like cells successfully, and possess the functional characterization of hepatocyte. PAS method indicating glycogen deposition was positive(prunosus particles within cytoplasm) on day 14 after inducing both in the labeled-induced group and unlabeled-induced group, and on day 21, the cells were strongly positive for glycogen of the two groups. After inducing 21 days, the results of PAS staining together with Prussian blue staining were as following:glycogen deposition and intracellular iron particles were found on the labeled-induced group; glycogen deposition was on the unlabeled-induced group; intracellular iron uptake was on the labeled-uninduced group; and none was found on the uninduced-unlabeled group. Undifferentiated cells were domenstrated as negative at immunocytochemistry stain for albumin, but positive staining on day 14 and 21 in the labeled-induced group and unlabeled-induced group. The two groups both express the ALB on 14d and 21d by RT-PCR, which expressed higher on 21d, and there is no significant difference between the two groups on day 14 and 21, respectively (P>0.05). These results indicated intracellular magnetic labeling with SPIO did not affect the capability of ADSCs to differentiate into hepatocyte-like cells.ConclusionOur initial results showed that SPIO could effectively label stem cells with the need for secondary TAs and it did not alter the cell viabitly and differential potential into hepatolike-cells of the labeled stem cells.