Selection and amplification of the liver stem cell subset from rat bone marrow cells with a medium containing cholestatic serum in vitro

AIM: To explore the feasibility of direct separat and selective enlargement of the bone marrow-derived liver stem cells (BDLSC) from bone marrow cells with a culture system containing cholestatic serum in vitro. METHODS: Bone marrow cells of rats were cultured with selective media containing 2%, 5%, 7% and 10% cholestatic rat serum, respectively. The BDLSC were then induced to proliferate with the addition of hepatocyte growth factor (HGF) on the firth day. BDLSC were characterized using immunocytochemistry and RT-PCR for lineage markers, glycogen staining and urea synthetic assay for functions 2 weeks later. RESULTS: Bone marrow cells were unble to form colony in the presence of 2% cholestatic serum and apopotosis appeared gradually in 7% or 10% cholestatic serum. The BDLSC survived in the medium containing 5% cholestatic serum while the other types of cells did not. The survival cells proliferated with a high speed during the second week and then formed hepatocyte-like colony-forming units (H-CFU). Cells in the H-CFU expressed the characteristic proteins of fetal hepatocytes. Furthermore, they had glycogen storage and urea synthesis functions, two of the critical features of hepatocytes. CONCLUSION: The selective micro-environment effectively selected BDLSC from the bone marrow cell, and will be a new way to provide an abundant source of donor hepatocytes for clinical cell therapy.     

Hepatic differentiation of mesenchymal stem cells derived from rats in three-dimensional microenvironment formed by hanging drops

AIM: To establish an optimal differentiated three-dimensional microenvironment formed by hanging drops for the high efficiency of hepatic differentiation from rat mesenchymal stem cells(rMSCs).METHODS: rMSCs were cultured in the hanging drops, which provided a three-dimensional microenvironment, for 21 days in the presence of hepatocyte growth factor(HGF, 20 μg/L). The expression of albumin(ALB), alpha-fetoprotein(AFP) and cytokeratin-18(CK-18) was detected by RT-PCR and immunofluorescence staining at 7th, 14th and 21st days. The secretion of albumin in the culture supernatants was measured by ELISA. RESULTS: rMSCs were aggregated in spheroid with a tubiform medium altitude in the center. In rMSCs cultured in the hanging drops with HGF, the expression of albumin, AFP and CK-18 was all detectable by RT-PCR and immunofluorescence staining at 7th day. The production of albumin in the cells cultured in the hanging drops with HGF was 50.25±5.32, 55.03±7.45 and 54.92±3.18(ng·dish^-1·d^-1) at 7th, 14th and 21st days, respectively, significantly higher than that in the cells in the plate cultivation with or without HGF induction at corresponding time points(P<0.01).CONCLUSION: In the presence of HGF, rMSCs are induced to differentiate into hepatocyte-like cells cultured in the hanging drops, where the three-dimensional spheroidal cultures are promising microenvironment for hepatic transdifferentiation of MSCs.     

Sodium butyrate induces mouse embryonic stem cells to differentiate into hepatocytes in vitro

AIM:To explore mechanism by which sodium butyrate induces mouse embryonic stem cells (ES) to differentiate into hepatocytes in vitro. METHODS:E14 mouse ES cells were cultivated in a routine way, and then cultivated in suspension to form embryonic bodies (EBs). EBs were transferred into 6-well culture dishes and 3 mmol/L sodium butyrate was added into the culture medium. Morphological changes were investigated by phase contrast microscopy. α-fetoprotein (AFP), albumin (ALB) and cytokeratin 18 (CK18) were examined by immunofluorescence staining. AFP, ALB, α1-antitrypsin (AAT) and TTR mRNA were assayed by RT-PCR. Proportion of ALB positive cells was analyzed by flow cytometry. Periodic acid Schiff (PAS) reaction and indocyanine green (ICG) uptake assay were performed to assess the characteristic hepatocyte function of the differentiated cells. RESULTS:In the presence of sodium butyrate, parts of ES cells differentiated into a population with epithelial morphology similar to mouse hepatocytes. AFP and TTR mRNA expression were observed at 7 d, and ALB and AAT mRNA expressed at 14 d. Hepatocytes specific markers, ALB, AFP and CK18 were positive expression in immunofluorescence staining at 14 d. PAS reaction and ICG uptake were positive for the hepatocyte-like cells. CONCLUSION:Mouse ES cells can be induced into hepatocyte-like cells by sodium butyrate efficiently, and these ES cells-derived hepatocytes possess characteristic hepatocytic function.     

Comparison of different conditions inducing embryonic stem cells

AIM: To evaluate the different conditions inducing mouse embryonic stem cells (ESC) in vitro to differentiate into cardiomyocytes. METHODS: BRL conditioned medium was used to promote the growth of ESC and maintain them in an undifferentiated state. During the inducing process, retinoic acid (RA), DMSO, activin-A and TGF-β₁ were used as inducing reagents, and made up six kinds of differentiating medium. Then a three-step method inducing ESC cultured in hanging drops, in suspension and in plating was used to induce the differentiation of ESC. RESULTS: ESC were induced in vitro to differentiate into cardiomyocytes. Of all groups, the highest differentiating rate was observed in the group induced by activin-A (20 μg/L) and TGF-β₁ (2 μg/L). CONCLUSION: The inducing conditions including activin-A (20 μg/L) and TGF-β₁ (2 μg/L) is very valuable in inducing ESC differentiation into cardiomyocytes.     

miRNA-122 promotes mouse embryonic stem cell-derived hepatic precursor cells to differentiate into hepatocytes

AIM：To investigate whether miRNA-122 (miR-122) promotes the differentiation of mouse embryonic stem cell (ESC)-derived hepatic precursor cells (HPCs) into hepatocytes. METHODS：Mouse ESCs were initially induced to differentiate into HPCs by stimulating with fibroblast growth factor 4 (FGF-4), sodium butyrate and dexamethasone (Dex) sequentially. Then a recombinant adenovirus expressing vector pAV.Ex1d-CMV>miR-122/IRES/eGFP was constructed by Gateway technology and transfected into the mouse ESC-derived HPCs 9 d after induction, so as to gain the cells with stable high expression of miR-122. The morphological changes of transfected cells were observed under inverted phase-contrast microscope. The liver-specific gene expression levels were detected by real-time RT-PCR. The liver-specific protein expression levels were also detected by immunofluorescence method. The liver functions were assessed by indocyanine green (ICG) uptake experiment, glycogen staining and urea synthesis function test. RESULTS：The mouse ESCs were successfully induced into HPCs by stimulating with FGF-4, sodium butyrate and Dex sequentially. At 6 d after transfection of miR-122, the morphology of the cells was closer to the mature hepatocytes. The mRNA levels of liver-specific genes such as albumin (ALB), transthyretion, α1-antitrypsin, glucose-6-phosphatase, cytokeration 8, cholesterol 7α-hydroxylase and cytochrome P450 3A4 were up-regulated. The expression levels of liver-specific proteins such as ALB and cytokeratin 18 were increased, while alpha-fetoprotein was decreased. The results of ICG uptake experiment, glycogen staining and urea synthesis function test indicated that the hepatocyte functions were strengthened as compared with control group. CONCLUSION：The combination of FGF-4, sodium butyrate and Dex successfully induces the mouse ESCs into HPCs. Over-expression of miR-122 effectively promotes the differentiation and maturation of mouse HPCs.     

Differentiation of rat bone marrow mesenchymal stem cells expressing hepatocyte growth factor into hepatocyte-like cells

AIM:To observe the hepatic differentiated efficiency of rat bone marrow mesenchymal stem cells (rMSCs) expressing hepatocyte growth factor (HGF) in three-dimensional microenvironment formed by hanging drop. METHODS:rMSCs were isolated and cultured in vitro, and flow cytometry was used to detect the expression of CD44, CD90, CD34 and CD45. Recombinant retrovirus carrying cDNA of human HGF (pLNCX2-hHGF) was constructed and infected with rMSCs (hHGF-rMSCs). hHGF expression in hHGF-rMSCs was detected by RT-PCR, immunofluorescence staining and ELISA. hHGF-rMSCs were cultured in hanging drop for 21 days. The expression of albumin (ALB),cytokeratin-18 (AFP) and alpha fetoprotein (CK-18) were detected by RT-qPCR and immunofluorescence staining in the 7th, 14th and 21st day, respectively. The secretion of albumin in cultured supernatant was measured by ELISA. RESULTS:CD44 and CD90 were highly expressed in the third generation of rMSCs, but CD34 and CD45 were hardly expressed. The expression of hHGF at mRNA and protein level were all detectable in the hHGF-rMSCs, and the secretion in the cultured supernatant was about (123.71±8.81) μg/L in a period of 21 days. In the hHGF-rMSCs, ALB, AFP and CK-18 were highly expressed at mRNA level from the 7th to the 21st day, and there were significant differences compared with rMSCs at the same time point (P<0.01). The results of immunofluorescence staining showed that the protein expression of AFP was negative on day 7 and 14, and positive on day 21; while the protein expression of ALB and CK-18 was positive on day 7 and lasted until day 21. ALB was positively observed in the culture supernatant of hHGF-rMSCs from 7th to 21st day measured by ELISA, and there was significant difference between the hHGF-rMSCs and rMSCs (P<0.01). CONCLUSION:hHGF transduced-rMSCs can be induced to differentiate into hepatocyte-like cells after cultured in hanging drop which provides a three-dimensional microenvironment. All these results might help to provide new seed cells for cell therapy of clinical liver diseases and in vitro bioartificial liver.     

Bone marrow mesenchymal stem cells are induced to differentiate into cardiomyocytes by hepatocyte growth factor in vitro

AIM: To explore a new method of hepatocyte growth factor (HGF) inducing bone marrow mesenchymal stem cells (MSC) to differentiate into cardiomyocytes. METHODS: Bone marrow MSC was cultured with DMEM media (10% fetal calf serum) 4-6 passages, and induced by HGF (10 μg/L) for 30 d. Automatical beating of the differentiated cells was observed daily with transverse microscopy, or under condition of 0.1% isoproterenol or cal-cium-deprived incubation. Specific cardiac myosin in the cells was indentified by immunochemistry. RESULTS: At 14-20 d of differentiation, bone marrow mesenchymal stem cells formed clones, in 10%-50% of which spontaneous beating cell-mass had come to continuously exist. Isoproterenol increased the beating rate and calcium-deprived media inhibited the beating. The cells were identified to be cardiomyocytes by expression of cardiac myosin heavy chain. CONCLUSION: HGF may induce bone marrow mesenchymal stem cells into cardiomyocytes with high efficiency, but the differentiating pathway of stem cells remains to be further studied.     

Human mesenchymal stem cells differentiate into neuron-like cells with Tanshinone II A

AIM:To investigate the differentiation from human mesenchymal stem cells(hMSC) into neuron-like cells with Tanshinone II A.METHODS:hMSC were separated from rib marrow with Ficoll-Paque reagent and expanded in culture medium. To detect the surface antigens, the labeled cells were analysed on a FACScan flow cytometer to determine the effect of the capacity of proliferation and differentiation of the mesenchymal stem cells with FGF-2. hMSC were induced to differentiate into neurons with DMEM Tanshinone II A. Neuron-specific enolase(NSE), neurofilament(NF), Nestin, glial fibrillary acaidic protein(GFAP) were detected by immunohistochemistry.RESULTS:hMSC were expanded as undifferentiated cells in culture for more than 15 passages. The isolated cultured MSC comprised a single phenotypic population and displayed a fibroblast-like morphology. These expanded attached MSC were uniformly positive for CD29, CD44, CD90, CD105, CD166 and didn't express CD11a, CD14, CD34, CD38, CD45, CD80, CD86. FGF-2 have special effect on low denisity MSCs. Simple methods with Tanshinone II A induced hMSC to exhibit a neuronal phenotype, expressing NSE, NF-M, Nestin at 5 hours. But the neuron-like cells didn't express the glial astrocyte marker GFAP.CONCLUSION:hMSC can be induced to differentiate into neurons with Tanshinone II A.     

A study of inductive factors of embryonic stem cells differentiating into cardiac myocytes in vitro

AIM: To study cellular and molecular mechanisms of cardiac development associated genes expression and its function during early stage cardiomyogenesis. METHODS: (1) Mouse embryonic stem cells (ESC) line D3 culture.(2) Inductive culture of ESC differentiated into cardiac myocytes in vitro. (3) Identification of ESC-derived cardiac myocytes: RNA isolation; synthesis of specific primer and RT-PCR; Label of RT-PCR products with dATP as probes, purifyed by sephadex G-50 columns, determined the yield of DNA. RNA dot hybridization. RESULTS: 80% of ESC differentiated into cardiomyocytes by improved conditional medium. Cardiomyocytes contracted in a synchronous manner. The results of RT-PCR and RNA blot showed that cardiac genes were expressed abundantly and specifically during the early cardiomyogenesis. CONCLUSIONS: ESC were able to be differentiate into cardiomyocytes. Different concentrations and components of RA, DMSO and FCS affected ESC cardiomyogenesis in vitro. The optimal result obtained was from the conditional medium, a mixturce of 2 nmol/L retinoic acid (RA), 0.6% dimethyl sulfoxide (DMSO) and 20% fetal calf serum (FCS).     

Hepatocyte growth factor enhances the neural differentiation from human embryonic stem cells

AIM: To investigate the possibility that hepatocyte growth factor (HGF) directly induces differentiation of human embryonic stem cells (hESCs) into neural progenitors (NPs). METHODS: hESCs colonies were induced to form the embryoid body (EB). Four-day-old EBs were randomly divided into 4 groups: control group (EBs were cultured in neural induction medium); G5 supplement group (EBs were cultured in neural induction medium supplied with G5 supplement); HGF group (EBs were cultured in neural induction medium supplied with 10 mg/L HGF), and HGF+G5 group (EBs were cultured in neural induction medium supplied with 10 mg/L HGF and G5 supplement). After induced in suspension system for 7 days, EBs with various treatments were cultured in poly-D-lysine/laminin-coated plates for 7-10 days for selection of NPs. NPs were gathered by 0.3 g/L dispase treatment and characterized by immunofluorescence staining. The percentages of the nestin+ cells in NPs in various groups were detected by fluorescent activated cell sorter (FACS). The multipotency of NPs was determined by immunofluorescence staining after the NPs were cultured without G5 and HGF for 7 days. The expression of region markers of neural progenitors treated with sonic hedgehog (Shh) protein (one of the neural inductive signals), was detected by RT-PCR. RESULTS: HGF+G5 supplement induced hESCs differentiation into neural progenitors. Immunofluorescence staining indicated that NPs differentiated from hESCs expressed NP markers including nestin, Pax6 and musashi-1. FACS data showed that the proportion of nestin positive cells in HGF+G5 supplement group (87.3%±3.9%) was the highest in all treatment groups. The time of HGF and G5 supplement treatment was important to differentiate into NPs, the maximal effect was observed at 7th day. After treated with Shh, the expression of ventral forebrain/hindbrain marker genes (Nkk2.1, and Nkk2.2) and hindbrain progenitor marker gene Gbx2 in NPs were upregulated, while the forebrain progenitor marker genes Otx2 and Bf1 were downregulated. CONCLUSION: The neural induction system containing HGF and G5 supplement effectively induces the differentiation of hESCs into NPs, which might be a potent model for investigating the mechanism of neural development and differentiation.     

Primary study on the differentiation mechanism of embryonic stem cells to epidermal like cells induced by human amnion

AIM: To investigate the mechanism by which human amnion induced mouse embryonic stem (ES) cells to differentiate into epidermal like cells. METHODS: ES- BALB/ c cells were cocultured with human amnionintranswells for 4 - 5 days , andthose cultured alone without amnion were taken as control group. The morphological differentiation were observed . The committed differentiation of EScells into epidermal like cells were detected by integrin-β₁ , CK19 , CK15 andinvolucrin immunohistochemistry , respectively .RESULTS: After 4-5 days of coculture, ES cells differentiated into single layer of epidermal like cells, fitted tightly, with polyhedral in shape. The immunohistochemical staining results showed that, most of the cells were integin-β₁ positive, only a few cells were CK19 and CK15 positively stained. Most of the cells in control group died, the survived ones were different in morphological shapes, and no integrin-β₁, CK19 and CK15 positive cells were found. CONCLUSION: Soluble substances secreted by human amnion may play an important role in inducing the differentiation of mouse ES cells into epidermal like cells.     

A method of inducing mouse embryonic stem cells cultured in vitro to differentiate into cardiomyocytes

AIM:To evaluate the method of inducing mouse embryonic stem (ES) cells in vitro to differentiate into cardiomyocytes without using any chemical reagents.METHODS:BRL conditioned medium was used to promote the growth of ES cells and maintain them in an undifferentiated state before the experiment of differentiation. Then a three-step method including ES cell culture in hanging drops and in suspending was used to induce the differentiation of ES cells. RESULTS:Rhythmically contracting cells were observed among differentiated cells, which were proved to be cardiomyocytes with electron microscope and immunocytochemistry. CONCLUSION:A simple and economical method was established to induce mouse ES cells cultured in vitro to differentiate into cardiomyocytes without using any chemical reagents.     

Direct differentiation of embryonic stem cells into neural cells without embryonic body culture period in vitro

AIM:To investigate the feasibility and effect of directly differentiation of embryonic stem cells (ESC) into neural cells induced by retinoid acid (RA) without embryonic body (EB) culture period in vitro. METHODS:ESC were digested and divided into 4 groups:group A and B were undergone EB culturing. After that, cells in group A were induced by RA, cells in group B were differentiated spontaneously, cells in group C were committedly induced by RA directly without EB culturing, and cells in group D were differentiated spontaneously without EB period. Morphologic changes were observed under inverted microscope and scanning electron microscope. MAP-2 and GFAP were detected by immunocytochemistry and flow cytometry after differentiated for 9 days. RESULTS:In groups A or C, neuron-like cells increased gradually, forming neural network. At the 9th day, a large part of cells in these groups were MAP-2 positive cells, and the positive rate was higher than that in groups B or D (P<0.01). Groups B and D were almost epithelial-like cells. At the 9th day, GFAP positive cells were predominant. The rate of GFAP staining cells in groups A or C was significantly lower than that in groups B or D (P<0.01). There was no significant difference of MAP-2 or GFAP positive rates between group A and C, nor between group B and D (P>0.05). CONCLUSION:ESC was directly induced into neural cells by RA without EB culture period in vitro. This modified method has the same effect as the traditional RA 4－/4＋ assay and can replace the traditional method.      

Rat mesenchymal stem cells differentiate into neuron-like cells with adrenaline hormones

AIM: To investigate the differentiation from rat mesenchymal stem cells (rMSC) into neuron-like cells. METHODS:rMSC were separated from femur marrow and expanded in L-DMEM culture medium supplemented with 10% FSC. rMSC were induced to differentiate into neurons with L-DMEM/adrenaline,L-DMEM/noradrenaline and L-DMEM/isoprenaline, respectively. Meanwhile, rMSC were cultured in L-DMEM in control group. Nestin, neuron-specific enclose (NSE), glial fibrillary acidic protein (GFAP) were detected by immunocytochemistry. RESULTS: rMSC were expanded as undifferentiated cells in culture from 5 to 22 passages, indicating their differentiated capacity. Simple method induced rMSC to exhibit a neuronal phenotype, expressing positive NSE,nestin, and GFAP, at 5 hours in all group. The undifferentiating cells (control group 53.1%±4.3%), and differentiating cells (treated group: adrenaline 74.7%±2.6%; noradrenaline 75.9%±2.4%; isoprenaline 72.1%±4.4%), expressed characteristics of various neuronal cells, from 5 hours to 6 days. There were neuron-like cells in rMSC cultured in L-DMEM/10%FBS from 7 to 13 passage(66.5%±6.4%). CONCLUSION: It suggests that rat neural stem cells (rNSC) exist in bone marrow, rMSC can be differentiated into various neural cells with adrenaline hormones in vitro.     

Functional hepatic differentiation from mouse induced pluripotent stem cells in vitro

AIM: To directly differentiate the induced pluripotent stem cells (IPS cells) into hepatocytes in chemically-defined, monolayer conditions in vitro. METHODS: Based on the knowledge of mammalian hepatic development, we differentiated the IPS cells into hepatic lineage efficiently. The present study involves stepwise transition of IPS cells through a series of media designs to first specify cells towards anterior definitive endoderm, and secondly, to induce hepatic maturation. RESULTS: The IPS cells recapitulated the development of liver in vivo at RNA and protein level. Real-time PCR analysis was performed on cell preparations at various times during the culture process to determine the time course and degree to which the IPS cells differentiated toward a hepatocyte phenotype. Analysis included markers for endoderm-specific gene expression (Sox17; FoxA2, CXCR4), hepatocyte-specific gene expression (albumin and AFP), and markers for undifferentiated IPS cells (OCT4 and Nanog). The endoderm-specific gene expression increased early while the expression of OCT4 and Nanog decreased and hepatocyte-specific gene expression progressively increased over the course of the differentiation program. The resultant hepatocyte-like cells(HLCs) showed phenotypic similarities with adult hepatocytes (expressing AFP and albumin) and additionally showed functionality expected to well-differentiated hepatocytes such as indocyanine green(ICG) uptake and release. The P450 activity of cytochromsome was also positive. CONCLUSION: We successfully construct the IPS cells which efficiently differentiate into hepatocytes and recapitulate the hepatocytic development in vivo. The reproducible and efficient generation of HLCs from IPS cells represents a critical step towards the ultimate goal of producing functional HLCs for clinical use including cell transplantation or bioartificial liver support. Moreover, our system provide a valid way to study the liver development since it is monolayer, serum-free and mimic the in vivo liver development.     

Directed differentiation of embryonic stem cells into epithelial cells by co-culture with human laryngeal epithelial cell conditioned medium

AIM:To explore the culture and inductive condition for the directed differentiation of embryonic stem cells into epithelial cells in human laryngeal epithelial cell conditioned medium.METHODS:E14 murine stem cells were induced into embryoid body in vitro first,then the cells of embryoid body were cultured in the induction system containing human laryngeal epithelial conditioned medium and epithelial cell growth factors.On day 14 of culture,samples were detected for the cytokeratins 4,14,19 expressions at protein level.Morphology of the cells was also observed under microscope.RESULTS:During the process of epithelial induction,cells with epithelial cell morphology were seen under contrast microscope.Immunofluorescent assay showed that cytokeratins 4,14,and 19 were all expressed at protein level in differentiated cell.CONCLUSION:This study suggests that epithelial cells could be induced from the directed differentiation of embryonic stem cells in the conditioned medium of human laryngeal epithelial cell cultures.This work has provided basic data for artificial laryngeal epithelia preparation.     

In vitro differentiation of exocrine pancreatic cells from mouse embryonic stem cells

AIM: To explore the effects of sodium butyrate, activin A and dexamthasone on inducing mouse embryonic stem (ES) cells to differentiate into exocrine pancreatic cells in vitro. METHODS: E14 mouse ES cells were cultured in suspension to form embryonic bodies (EBs). The EBs were cultured with differentiating medium containing different concentrations of sodium butyrate, and the spontaneously differentiated ES cells were used as control. Exocrine pancreatic genes such as amylase, chymotrypsinogen, elastase 1, elastase 2 and carboxypeptidase were detected by RT-PCR at different time points to determine the optimal concentration and exposure time of sodium butyrate. Furthermore, activin A or dexamthasone was also used to explore the effects on exocrine differentiation. After that, the combination of sodium butyrate, activin A and dexamthasone was used to promote the differentiation of exocrine pancreatic cells from ES cells. During the differentiation course, the gene expressions of amylase, chymotrypsinogen, elastase 1, elastase 2 and carboxypeptidase were detected by RT-PCR. Morphological changes were investigated by phase contrast microscopy. Amylase expression was examined by immunofluorescence staining. RESULTS: Exocrine pancreatic gene expressions such as amylase, chymotrypsinogen, elastase 1, elastase 2 and carboxypeptidase were detected in spontaneously differentiated EBs. A relatively lower concentration of sodium butyrate with a shorter exposure time significantly promoted those above gene expressions as compared to that of spontaneously differentiated EBs. Activin A and dexamethasone induced upregulation of exocrine gene expression. The combination of activin A, sodium butyrate and dexamethasone significantly enhanced the mRNA levels of amylase, chymotrypsinogen, elastase 1, elastase 2 and carboxypeptidase. Under the treatment of activin A, sodium butyrate and dexamethasone, differentiated cells were polygonal in shape with large, round, and center-situated nuclei. According to the observation of immunofluorescence staining, amylase was positive expressed at the final stage. CONCLUSION: These data indicate that exocrine pancreatic differentiation of ES cells is induced by sodium butyrate, activin A and dexamethasone. The combination of pancreatic inducing factors improves the differentiating efficiency.     

The preliminary study on differentiation of mesenchymal stem cells into corneal epithelium like cells

AIM: To observe the differentiation and development of human mesenchymal stem cells (MSCs) transplanted onto corneal stroma of rabbits and investigate the feasibility of MSCs differentiated into corneal epithelium like cells.METHODS: 24 New Zealand albino rabbits were randomly divided into 2 groups: The human MSCs combined with amniotic membrane were transplanted onto the experimental animals, and the controls were transplanted with the amniotic membrane only. The MSCs were cultured on preserved human amniotic membrane for 4 days and labeled with 5’-Bromo-2’-deoxyuridine (BrdU), then the cells were transplanted onto the surface of the corneal stroma of the rabbits. The eyeballs were taken off after 1, 2, 3, 4, 6 and 8 weeks. The growth and differentiation of human MSCs were observed by histopathological and immunohistochemical examination.RESULTS: When the MSCs cultured on amniotic membrane were transplanted onto the surface of the corneal stroma of rabbits, the corneal epithelium were positive in CK3/CK12 staining and negative in CK 13 staining, revealed by immunohistochemical examination. The BrdU positive cells in the reconstructive corneal epithelium were found and showed positive in CK3/CK12 staining. CONCLUSION: After transplanted onto the corneal stroma of rabbits with human amniotic membrane, the MSCs survive, proliferate and differentiate into corneal epithelium like cells.     

Supportive and expansive effects of aorta-gonad-mesonephros (AGM)-derived stromal cells on hematopoietic stem in vitro

AIM: To explore the supportive and expansive effects of aorta-gonad-mesonephros(AGM) region derived stromal cells on hematopoietic stem cells (HSC) in vitro.METHODS: The murine stromal cells were separated and cultured from AGM region of a 11 day postcoitum (dpc) mouse embryo and 6 week mouse. After identification by Wrights staining and flow cytometry, the stromal cells were co-cultured with the embryonic stem cell(ESC)-derived, cytokine-induced HSCs, and the maintenance and expansion of HSCs were evaluated by detecting CD34+，CD34+Sca-1+cells with flow cytometry.Blast colony-forming cell (BL-CFC) and high proliferative potential colony-forming cells(HPP-CFC) were determined by semi-solid medium clonal culture.RESULTS: AGM-derived and bone marrow(BM)-derived stromal cells were similar in morphology and phenotype, and had common character of stromal cells. Supported by AGM stromal cells or by BM stromal cells, more primitive progenitor cells HPP-CFC were expanded, but BL-CFC expansion was only detected in AGM-derived stromal cells. In the supporting of BM stromal cells CD34+ hematopoietic stem/progenitor cells were expanded 3-4 times, but no significant expansion in CD34+Sca-1+ cells was observed. While in the supporting of AGM stromal cells, both CD34+ hematopoietic stem/progenitor cells and CD34+Sca-1+ cells were expanded significantly from 4 to 5 times, respectively (P<0.05).CONCLUSION: AGM-derived stromal cells significantly support the expansion of HSC, and also maintain the self-renewal activity and multi-lineage differentiation of HSC in vitro.     

Embryonic stem cells transplant into subretinal space of C3B mouse

AIM:To study the differentiation of embryonic stem cells (ESC) at the subretinal space of C3B mouse whose retina are of normal structure. METHODS:Embryonic bodies (EB) were gained when embryonic stem cells were propagated 1 generation after anabiosis. The digested EB were transplanted into subretinal space of C3B mouse. The eyes were analysed by photo microscopy, electric microscopy and immune assay at 1st week, 3rd week and 2nd month after injection. RESULTS:The thickened retina appeared on the place of injection at 1st week. Teratoma formation was observed in 3 transplant recipients at 3rd week and 2nd month. The ratio of forming tumor was 25% in all eyes received cell transplant. Eye atrophy or scar at injection position was seen in other eyes. The nucleus of tumour cells had apparent strange type under electrical microscopy. The immune assay showed that part of the tumour were microtubule-associated protein 2 (MAP-2) positive;part of the tumour were glial fibrillary acid protein (GFAP) positive;a little cells were nestin positive. CONCLUSION:After transplantation into the subretinal space, ESC were not incorporated into retina or differentiated into retinal cells. The teratoma was formed in part of eyes. The security of application for ESC in eye was considerable.