Investigating the function of combining induced rat monocytes-derived bone marrow-haemopoietic stem cell (rat BM-HSCs) with LPS and rat bone marrow-mesenchymal stem cell (rat BM-MSCs) was to analyze the acceleration of homing process mechanism in injured pancreas. Mononucleated stem cells were isolated from aspirated whole rat BM using ficoll and cultured in α-MEM complete growth medium in 10 cm petridish. After two days, adherent cells after washing twice in petridish were added α-MEM growth medium and then mesenchymal cells were characterized using CD105 marker in third passage and labeled PKH26. Then haemopoietic stem cells (HSCs) were isolated with magnetic beads CD34+ and differentiated in vitro, and then induced monocytes with LPS. Animal experiment used 28 male Wistar rats, and divided them into 4 groups. After transplantation combined, both cells between monocyte derived HSc (mHSCs) and rat BM-MSC were analyzed expression of pair box gen 4 (Pax4), pancreatic and duodenal homeobox (Pdx1), C-peptide using immunohistochemistry, then secretion of insulin and C-peptide analyzed using indirect ELISA. Results showed that the expressions of Pax4, Pdx1, C-peptide found in the surface membrane cell of pancreatic cell, and secreted C-peptide and insulin were shown significant (P < 0.05) in transplanted group 2, 3 and 4, but in group 3 were transplanted with combined cells more dominant than non-combined cells. Conclusions suggested that combining of induced monocytes-derived HSCs and rat BM-MSCs has accelerated homing MSCs into injured pancreatic tissue.
The role of MSCs in the modulation of the immune response, immune system activity, and the body’s response to inflammation and disease has been widely studied for many years, but homing mechanisms are still limited. MSCs both rely upon and co-create a network that facilitates constant communication between normal and damaged cells in the body [
In this study, the acceleration of homing mechanism using induced monocytes-derived HSCs CD34+ in injured pancreas was analyzed, because activated monocytes can express many kinds of molecule signaling like CRCX4 and TNF-α that can induce migration of rat BM-MSCs to the injured area. Monocyte chemo-attractant (MCP)-1 is typically expressed at sites of inflammation and can thus represent a model homing chemokine, because MSCs have expressed the MCP-1 receptor and chemokine receptor (CCR) 2 on the cell surface membrane cell [
Multiple approaches are now being investigated to generate insulin-producing cells in vitro either by genetic engineering of β-cells or by utilizing various β-cell precursor cells and stem/progenitor cells with the ability to grow in vitro and to differentiate into insulin-producing cells and ultimately into β-cells [
This experiment used male Wistar rats, weight 200 gram and age 3 mounths were obtained from Veterenary Farma, Surabaya, Indonesia who that undergoverment instituton has produced for vet vaccine, diagnostic and has breeding of some kinds animals laboratory. Four groups of 28 Wistar rats, first group for control and each of second to fourth groups were treated using aloxan injection in dose of 50 mg/kg intraperitoneally according to method of Purwati [
Whole bone marrow were aspirated about 3 ml then isolated using Ficoll 0.077 density (Invitrogen). After centrifugation by 1600 rpm, 30 min were isolated mononucleates cells from Buffy coat space and washed using growth medium serum free and followed centrifugation by 1600 rpm in 10 min. After that were cultured mononucleate cells in petridish 10 cm and growing in complete medium (α-MEM) (Invitrogen) according to modified method of Wolf [
This method was used to characterize of phenotype rat Bone-Marrow derived Mesenchymal Stem Cell (rBM- MSC) specific CD105 and CD34+. Cells were fixed in ice-cold methanol at ?20˚C. Following two washes with PBS, the cells were blocked for 1 hour in PBS containing 1% bovine serum albumin (Sigma-Aldrich Corp) and 0.1% Triton X-100 (Sigma-Aldrich Corp) for 45 min and then incubated in the primary antibody (CD105, 1:100, Dako) for 2 hour at room temperature. After three washes with PBS, the cells were incubated in secondary antibody (fluorescein isothiocyanat-conjugated antibodies rabbit anti rat-Dako) for 1 hour, and after final wash, the cells were imaged using an Olympus inverted fluorescent microscope.
Flow cytometric analysis for phenotype including isotype control, was performed according to manufactures. Freshly isolated cells were incubated for 15 min with erythrocyte lysis buffer at 41˚C then were cells washed with PBS, and incubated with Dil-ac-LDL (acLDL) (10 mg/ml) for 1 hour at 37˚C. Afterwards, cell were washed twice with plain medium, once with PBS, and fixed with 4% paraformaldehyde (PFA), and then cell were centrifuged at 1600 rpm for 10 men and resuspended in PBS containing 0.2% FCS and 2 mM EDTA. For incubation with primary anti rat CD105-FITC cell were also fixed with 4% PFA, resuspended in PBS containing 0.2% FCS and 2 mM EDTA, and maintained at 41˚C for 30 min. After washing and incubation with secondary FITC-conjugate antibody was performed for 30 men at 41˚C with sample. Labeled cells were analyzed by flowcytometry according to the guide of BD (BD Biosciences, San Jose, CA) and cellQuest pro analysis. The percentage of positively stained cells was determined after correction for the percentage of cell reacting with the secondary FITC antibody.
Whole rat bone marrows were isolated using CD34+ magnetic beads (Invitrogen). After centrifugation by 1600 rpm in refrigerator centrifuge (Backman) was supernatant removed and resuspended in buffer elusion reagent (invitrogen) then repeated centrifugation by 1600 rpm at 15 min. After removing of supernatant were cells resuspended using complete medium α-MEM and plated in petridish, and for immunotyphing using flow cytometry according to modified method of Tarja and Sharkis [
Cell sections of pancreatic tissue were fixed with 4% paraformaldehyde (Sigma) for 10 min. Immunohistochemistry was performed as previously described by Shuang-zhi [
Microplates (96-well, Nunc) were coated with rat sera from each group overnight at 4˚C, washed with PBS containing Tween 20 (PBST) and blocked for 1 h at room temperature with 1% BSA. After three washes with PBST, the primary mAb anti C-peptide, insulin goat anti-rat were diluted appropriately in PBS 1:200 and incubated on the plates for 1 h at 37˚C. After three washes with PBST, horseradish peroxidase-conjugated anti-rat immunoglobulin was diluted in PBS 1:1500 and added to the wells. Following incubation for 45 min at 37˚C, the plates were washed three times with PBST, and ortho-phenylenediamine (OPD) substrate (Sigma) was added. The plates were incubated 10 min in the dark. After the addition of 1 M HCl, the plate was read on an ELISA reader (BioRad) with the 450 nm wavelength. This method was modified from Tanaka [
Experimental data were presented as mean ± SD. Statistical calculations were performed using SPSS13.0 (SPSS Inc., USA). For statistical analyses, were used the Student’s t-test to compare data from normal donors with that obtained from injured pancreas and used a non-parametric test to compare the expression of pancreatic-specific markers. Statistical significance was defined as p < 0.05.
Rat bone marrow mesenchymal stem cells (rat BM-MSCs) were demonstrated previously by Purwati [
Bone marrow mesenchymal stem cell (BM-MSCs) has many kinds of markers like CD73, CD90, CD105 [
In these cases have been enough of MSCs count expressed CD105, these results indicate homogeneity and viability of BM-MSCs high. Although this marker was used mostly for proliferation and differentiation cells into osteoblast, chondroblast [
The results showed in
The data in
once of migration factor during homing process. Although in generally showed that expression of CXCR4 relative highest 10% than TIR.
Data in
and extra cellular matric have most important role. Also expression of Pdx1, Pax4 and C-peptide have shown activated β islet cells as transcription factor had good function in the homing as early in healing mechanism. The others hand, the expression of C-peptide in injured pancreatic like
Mesenchymal stem cell (MSC) is multipotent stem cell, that has a plasticity properties, and also have many kinds of protein as markers or signal expressed on the surface membrane cell have important role in communication between cell. CD105 is relative stable markers of MSC, because many kinds of markers can be used for detection of MSCs like CD90, CD73 and cells did not express CD34, CD11b, CD19, CD45 and HLA-DR [
The other hand, we used CD34+ for isolation of haemopoietic stem cell from bone marrow. Because this marker was stable for mononucleat haemopoietic stem cell, and also were differentiated into monocyte, and characterized by monocyte (Mac1) markers. Induced monocyte derived HSCs (IM-HSCs) can express toll interleukine receptor (TIR) and CRCX4 like in
BM-MSCs have many kind of ligands expression and also IM-HSCs have many kinds of receptors with different function, but they were influenced high by microenvironment. This way the stem cell can derived into different type of desire cell. In this research, we used rat BM-MSCs with expression of CD105 positive 81.3% in
The primary goal of this study was to determine of the accelerated homing function by combining BM-MSCs and IM-HSCs, and characterized by expression of Pax4, Pdx1, and C-peptide on the surface membrane pancreatic cells.
Combining MSC and monocytes HSCs transplantation can increase activation of homing process than without combining stem cell. Other indicators showed TIR as signaling molecule, CXCR4 as adhesion molecule were increasing. The other meaning that both molecule especially monocytes HSCs can induce stem cells migration and adhesion into injured tissue. In order for MSCs to provide a pleiotropic immunomodulatory effect that is responsive to different stimulants such as cytokines and chemokines and targets different effectors cells such as T-cells, NK-cell and DCs, it seems reasonable for MSCs to employ both by direct and soluble mediators that coordinate for a multi-pronged approach to therapy [
Pax4 is positioned in the upper hierarchy among the β-cell transcription factors, and its expression in β-cell precursors results in the differentiation and maturation of β-cell. The results in
Pdx1 expression like in
C-peptide is produced by a series of enzymatic cleavage of the precursor molecule preproinsulin and proinsulin. Preproinsulin is a precursor of proinsulin, is produced in the endoplasmic reticulum of pancreatic β-cell in respon to elevated blood glucose levels [
In this research, pancreatic cells were injured with alloxan and then pancreatic cell was releasing stromal derived factor-1 (SDF-1). Alloxan would induce reactive oxygen species. The action of reactive oxygen species with a simultaneous massive increase in cytosolic calcium concentration causes rapid destruction of B cells [
Secretion of C-peptide, Insuline
Type | G1/plasebo | G2/BM-MSCs | G3/BM-MSCs and IM-HSCs | G4/Induced Monocyte HSCs |
---|---|---|---|---|
C-peptide ng/ml | 1.02 ± 0.05 | 0.4 ± 0.01 | 1.00 ± 0.05* | 0.30 ± 0.01 |
Insulin ng/ml | 0.49 ± 0.05 | 0.36 ± 0.05 | 0.42 ± 0.02* | 0.20 ± 0.01 |
*Statistical significance from serum rat injured pancreas after transplanted combined between BM-MSCs and IM-HSCs.
The chemokine SDF-1 are involved in cell migration, survival, and development of any cell types including hematopoeitic stem cells (HSC) and stromal stem cells (SSC) through CXCR4, during homeostasis CXCR4 highly expressed in HSC and low in SSC. In host defense and repair mechanism, SDF-1 and CXCR4 can recruitment of immature and maturing leukocytes from the bone marrow to damage organ like injured pancreatic cells. Homing stem cells in their niches regulate by the cross-talk between CXCR4 on hematopoietic cells and stromal cell-derived SDF-1 [
MSC have been shown to support hematopoiesis, it would be advantageous to co-transplant donor MSC with HSC to promote the rate of engraftment. Unfortunately, the transplant ability of marrow stromal elements remains controversial, with most studies showing that MSC, or stromal cells, have a limited capacity to reconstitute the marrow microenvironment. The fact that methods for isolation and amplification of MSC have been well studied and MSC home to BM with poor efficiency prompted us to find approaches to promote MSC homing and elucidate the mechanisms that guide homing of implanted MSC. Lately, CXCR4 was published to be expressed on the surface of MSC and BM stromal cells. The small proportion of MSC was expressed CXCR4, which contributed to their migration that capable of promoting migration to pancreatic islets in vitro. SDF-1 was reported could induce migration of human BM stromal cells in vitro and that CXCR4 might play a role in the engraftment of these cells in brain tissue of immunodeficient mice.
In this experiment we demonstrate for the first time that enforced surface expression of CXCR4, TIR and MAC-1 by inducing CD34+ with LPS was able to enhance in vivo homing on pancreatic islet. CXCR4 is known to be up regulated when human MSC are exposed to cytokines, including insulin-like growth factor-1, which is present in cell culture serum. Several studies now report that human MSC migrate in vitro in response to SDF-1, perhaps mediated by up regulation of intracellular CXCR4 molecules [
Beside MAC-1, LPS can induce hematopoietic stem cell expression of TIR/TLR4 through TRIF pathway [
Homing stem cell can accelerate using combining between MSC and HSC. MSC has many kinds of molecular signaling or ligands on the cell surface, but monocytes HSC has many kinds of molecular ligands activated by cell and others cell to migration or interaction intracellular, and finally the cell can be easy to attachment to the other cell, then proliferation and differentiation and can integrate with host tissue. TIR and CCXR4 have an important role in homing process as attractants of MSCs. The accelerated mechanism of homing stem cell in injured pancreatic was showed in
We thank to all researcher team at the Stem Cell Center, Institute of Tropical Disease Airlangga University Surabaya, East Java, Indonesia, Helen Susilowati, Eryk Hendrianto, Anas Prasetya Adi, Deya Karsari, Aristika Dinar Yanti, Nora Ertanti and Zahrotul Hasanah thank you for excellences of technical assistant.