Differentiated human adipose-derived stromal cells exhibit the phenotypic and functional characteristics of mature Schwann cells through a modified approach

Background aimsTissue engineering technology is a promising therapeutic strategy in peripheral nerve injury. Schwann cells (SCs) are deemed to be a vital component of cell-based nerve regeneration therapies. Many methods for producing SC-like cells derived from adipose-derived stromal cells (ADSCs) have been explored, but their phenotypic and functional characteristics remain unsatisfactory.MethodsWe investigated whether human ADSCs can be induced to differentiate into mature and stable SC-like cells with the addition of insulin, progestero``ne and glucocorticoids. The phenotypic and functional characteristics of new differentiated ADSCs (modified SC-like cells) were evaluated by real-time quantitative polymerase chain reaction, enzyme-linked immunosorbent assay and immunocytochemistry in vitro. Cells loaded into collagen sponge biomaterials were implanted around transected sciatic nerves with a 10-mm gap in vivo. The axon regrowth and functional recovery of the regenerated nerves were assessed by immunohistochemistry and Walking footprint analysis.ResultsAfter differentiation induction, the modified SC-like cells showed significantly up-regulated levels of S100B and P0 and enhanced proliferative and migratory capacities. In addition, the modified SC-like cells showed increased secretion of neurotrophic factors, and their functional characteristics were maintained for more than 3 weeks after removing the induction reagents. The modified SC-like cells exhibited significantly enhanced axon regrowth, myelination and functional recovery after sciatic nerve injury.ConclusionsOverall, the results suggest that this modified induction method can induce human ADSCs to differentiate into cells with the molecular and functional properties of mature SCs and increase the promotion of peripheral nerve regeneration.     

Schwann-like cell differentiation of rat adipose-derived stem cells by indirect co-culture with Schwann cells in vitro

Objectives: Schwann cell (SC) transplantation is a promising therapy for peripheral nerve transaction, however, clinical use of SCs is limited due to their very limited availability. Adipose‐derived stem cells (ADSCs) have been identified as an alternative source of adult stem cells in recent years. The aim of this study was to evaluate the feasibility of using ADSCs as a source of stem cells for differentiation into Schwann‐like cells by an indirect co‐culture approach, in vitro. Materials and methods: Multilineage differentiation potential of the obtained ADSCs was assayed by testing their ability to differentiate into osteoblasts and adipocytes. The ADSCs were co‐cultured with SCs to be induced into Schwann‐like cells through proximity, using a Millicell system. Expression of typical SC markers S‐100, GFAP and P75NTR of the treated ADSCs was determined by immunocytochemical staining, western blotting and RT‐PCR. Myelination capacity of the differentiated ADSCs (dADSCs) was evaluated in dADSC/dorsal root ganglia neuron (DRGN) co‐cultures. Results: The treated ADSCs adopted a spindle shaped‐like morphology after co‐cultured with SCs for 6 days. All results of immunocytochemical staining, western blotting and RT‐PCR showed that the treated cells expressed S‐100, GFAP and P75NTR, indications of differentiation. dADSCs could form Schwann‐like cell myelin in co‐culture with DRGNs. Undifferentiated ADSCs (uADSCs) did not form myelin compared to DRGNs cultured alone, but could produce neurite extension. Conclusions: These results demonstrate that this indirect co‐culture microenvironment could induce ADSCs to differentiate into Schwann‐like cells in vitro, which may be beneficial for treatment of peripheral nerve injuries in the near future.     

Rho-Associated Protein Kinases Play an Important Role in the Differentiation of Rat Adipose-Derived Stromal Cells into Cardiomyocytes In Vitro

Adipose-derived stromal cells (ADSCs) represent a readily available abundant supply of mesenchymal stem cells and have the ability to differentiate into cardiomyocytes in mice and human, making ADSCs a promising source of cardiomyocytes for transplantation. However, there has been no report of differentiation of rat ADSCs into cardiomyocytes. In addition, signaling pathways in the differentiation process from ADSCs to cardiomyocytes are unknown. In this study, we first demonstrated that rat ADSCs spontaneously differentiated into cardiomyocytes in vitro, when cultured on a complete medium formulation MethoCult GF M3534. These differentiated cells possessed cardiomyocyte phenotype and expressed cardiac markers. Moreover, these cells showed open excitation-contracting coupling and Ca²⁺ transient and contracted spontaneously. The role of Rho-associated protein kinases (ROCKs) in the differentiation process was then studied by using ROCK-specific inhibitor Y-27632 and ROCK siRNAs. These agents changed the arrangement of cytoskeleton and diminished appearance of cardiomyocyte phenotype, accompanied by inhibition of c-Jun N-terminal kinase (JNK) phosphorylation and promotion of Akt phosphorylation. Collectively, this is the first study to demonstrate that rat ADSCs could spontaneously differentiate into cardiomyocytes in vitro and ROCKs play an important role in the differentiation of ADSCs into beating cardiomyocytes in conjunction of the PI3K/Akt pathway and the JNK pathway.     

Immunohistological Localization of Endogenous Unlabeled Stem Cells in Wounded Skin

Various types of endogenous stem cells (SCs) participate in wound healing in the skin at different anatomical locations. SCs need to be identified through multiple markers, and this is usually performed using flow cytometry. However, immunohistological identification of endogenous stem cells in the skin at different anatomical locations by co-staining multiple SC markers has been seldom explored. We examined the immunohistological localization of four major types of SCs in wounded skin by co-staining for their multiple markers. Hematopoietic SCs were co-stained for Sca1 and CD45; mesenchymal SCs for Sca1, CD29, and CD106; adipose SCs for CD34, CD90, and CD105; and endothelial progenitor cells and their differentiated counterparts were co-stained for CD34, Tie2, and von Willebrand factor. We found Sca1⁺CD45⁺ SCs in the epidermis, dermis and hypodermis of wounded skin. Sca1⁺CD29⁺ and Sca1⁺CD106⁺ mesenchymal SCs, CD34⁺CD105⁺, CD34⁺CD90⁺, and CD90⁺CD105⁺ adipose SCs, as well as CD34⁺Tie2⁺ endothelial progenitor cells were also located in the epidermis, dermis, and hypodermis. This study demonstrates the feasibility of using immunohistological staining to determine the location of SCs in wounded skin and the intracellular distribution of their molecular markers.     

Oligoprogenitor Cells Derived from Spermatogonia Stem Cells Improve Remyelination in Demyelination Model

Embryonic stem (ES) like cells-derived testis represents a possible alternative to replace of neurons and glia. Here, we differentiated spermatogonia cells to oligoprogenitor (OP) like cells and transplanted them to demyelination model and assess their recovery potential in a demyelinated corpus callosum model in rats. ES like cells were differentiated to OP like cells using appropriate inducers and were transplanted in situ to demyelinated corpus callosum. Cell integration as well as demyelination extension and myelination intensity changes were evaluated using histologic studies and immunocytochemistry after 2 and 4 weeks post transplantation. Investigation of Nestin, NF68, Olig2, and NG2 by immunocytochemical technique indicated the differentiation of ES like cells to neuroprogenitor and oligodendrocyte like cells in each induction stage. Histologic findings showed a significant decrease in demyelination extension and a significant increase in remyelination intensity in cell transplanted groups. Also on the base of PLP expression, differentiation of transplanted cells was confirmed to myelinogenic cells using immunocytochemistry technique. We conclude that ES like cells derived from spermatogonia cells can be differentiated to OP like cells that can form myelin after transplantation into the demyelination model in rat, this represents recovery potential of spermatogonia cells which opens new window for cell therapeutic approaches using spermatogonial stem cells.     

Let-7a-5p regulated by lncRNA-MEG3 promotes functional differentiation to Schwann cells from adipose derived stem cells via directly inhibiting RBPJ-mediating Notch pathway

Schwann cells (SCs) have important roles in supporting and repairing peripheral neurons, and thus have great potential for nerve injury treatment. Adipose tissue-derived stem cells (ADSCs) can be reliably induced to differentiate into SCs. However, the underlying molecular mechanisms are unclear. We explored the roles of MEG3/let-7a-5p/RBPJ axis in the differentiation into SCs from ADSCs. Primary ADSCs were induced to differentiate into SCs by appropriate reagents. ELISA, immunostaining, Western blotting, and qRT-PCR were employed to examine levels of SC-markers such as S100, GFAP, SOX10, p75NTR, GAP43, MPZ, β-NGF, BDNF, and NCAM and let-7 family, MEG3, RBPJ, and Notch signaling related proteins. Dual luciferase assay and RNA immunoprecipitation were performed to validate interactions of let-7a-5p/RBPJ mRNA and MEG3/let-7a-5p. Cultured ADSCs could be induced to differentiate into functional SCs. Let-7a-5p and let-7d-5p were elevated during the differentiation while MEG3 and RBPJ/Notch-signaling were suppressed. Let-7a-5p mimics promoted ADSC differentiation into SCs and up-regulated the levels of SC-related markers including S100, GFAP, SOX10, p75NTR, GAP43, MPZ, β-NGF, and NCAM, while RBPJ or MEG3 overexpression retarded the differentiation and reduced those levels. Let-7a-5p directly targeted RBPJ and MEG3 disinhibited Notch-RBPJ signaling via sponging let-7a-5p. RBPJ overexpression reversed the acceleration of let-7a-5p mimics on SC differentiation while let-7a-5p mimics blocked MEG3-mediated suppression on SC differentiation. Let-7a-5p sponged by MEG3 promotes differentiation of ADSCs into SCs via suppressing Notch signaling by targeting RBPJ. These findings shed light on mechanisms underlying the differentiation of ADSCs to SCs and provide avenues to accelerate the process.

     

Differentiation potential of rabbit CD90-positive cells sorted from adipose-derived stem cells in vitro

To investigate the differentiation potential of purified CD90⁺ cells sorted from adipose-derived stem cells (ADSCs), CD90⁺ cells were sorted from rabbit ADSCs using flow cytometry. Then, cell expansion of CD90⁺ cells and unsorted ADSCs was observed using an inverted microscope. Furthermore, cell surface markers including CD40, CD105, and CD90 on CD90⁺ cells and unsorted ADSCs were quantified using flow cytometry. Additionally, multi-lineage differentiation ability between CD90⁺ cells and unsorted ADSCs was compared, and expression of adipocyte-related genes PPAR-r and CEBPA as well as stem cell-related gene SOX2 in CD90⁺ cells and unsorted ADSCs was determined using real-time quantitative PCR. We found that CD90⁺ cells had a stronger cell proliferation ability than unsorted ADSCs. CD90⁺ cells showed a stronger ability of osteoblast and chondrocyte differentiation than unsorted ADSCs and CD90^? cells, whereas the adipose differentiation ability of CD90⁺ cells was similar to that of ADSCs and CD90^? cells. CD14, CD105, and CD90 on CD90⁺ cells were expressed more highly than those on ADSCs. Additionally, the mRNA expression level of SOX2 in CD90⁺ cells was significantly higher than that in ADSCs, whereas the expression of PPAR-r and CEBPA was markedly lower than that in ADSCs. These results suggested that the purified CD90⁺ cells sorted from ADSCs exhibit a stronger differentiation potential than the unsorted ADSCs.     

Adipose-Derived Stem Cells Expressing the Neurogenin-2 Promote Functional Recovery After Spinal Cord Injury in Rat

Neurogenin2 (Ngn2) is a proneural gene that directs neuronal differentiation of progenitor cells during development. This study aimed to investigate whether the use of adipose-derived stem cells (ADSCs) over-expressing the Ngn2 transgene (Ngn2–ADSCs) could display the characteristics of neurogenic cells and improve functional recovery in an experimental rat model of SCI. ADSCs from rats were cultured and purified in vitro, followed by genetically modified with the Ngn2 gene. Forty-eight adult female Sprague–Dawley rats were randomly assigned to three groups: the control, ADSCs, and Ngn2–ADSCs groups. The hind-limb motor function of all rats was recorded using the Basso, Beattie, and Bresnahan locomotor rating scale for 8 weeks. Moreover, hematoxylineosin staining and immunohistochemistry were also performed. After neural induction, positive expression rate of NeuN in Ngn2–ADSCs group was upon 90 %. Following transplantation, a great number of ADSCs was found around the center of the injury spinal cord at 1 and 4 weeks, which improved retention of tissue at the lesion site. Ngn2–ADSCs differentiated into neurons, indicated by the expression of neuronal markers, NeuN and Tuj1. Additionally, transplantation of Ngn2–ADSCs upregulated the trophic factors (brain-derived neurotrophic factor and vascular endothelial growth factor), and inhibited the glial scar formation, which was indicated by immunohistochemistry with glial fibrillary acidic protein. Finally, Ngn2–ADSCs-treated animals showed the highest functional recovery among the three groups. These findings suggest that transplantation of Ngn2-overexpressed ADSCs promote the functional recovery from SCI, and improve the local microenvironment of injured cord in a more efficient way than that with ADSCs alone.     

Efficient generation of functional Schwann cells from adipose-derived stem cells in defined conditions

Schwann cells (SCs) are hitherto regarded as the most promising candidates for viable cell-based therapy to peripheral nervous system (PNS) injuries or degenerative diseases. However, the extreme drawbacks of transplanting autologous SCs for clinical applications still represent a significant bottleneck in neural regenerative medicine, mainly owing to the need of sacrificing a functional nerve to generate autologous SCs and the nature of slow expansion of the SCs. Thus, it is of great importance to establish an alternative cell system for the generation of sufficient SCs. Here, we demonstrated that adipose-derived stem cells (ADSCs) of rat robustly give rise to morphological, phenotypic and functional SCs using an optimized protocol. After undergoing a 3-week in vitro differentiation, almost all of treated ADSCs exhibited spindle shaped morphology similar to genuine SCs and expressed SC markers GFAP and S100. Most importantly, apart from acquisition of SC antigenic and biochemical features, the ADSC-derived SCs were functionally identical to native SCs as they possess a potential ability to form myelin, and secret nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF) and glia-derived neurotrophic factor (GDNF). The current study may provide an ideal strategy for harvesting sufficient SCs for cell-based treatment of various peripheral nerve injuries or disorders.     

Comparison of the Efficiencies of Three Neural Induction Protocols in Human Adipose Stromal Cells

The aim of this study was to compare the neural differentiation potential and the expression of neurotrophic factors (NTFs) in differentiated adipose-derived stem cells (ADSCs) using three established induction protocols, serum free (Protocol 1), chemical reagents (Protocol 2), and spontaneous (Protocol 3) protocols. Protocol 1 produced the highest percentage of mature neural-like cells (MAP2ab⁺). Protocol 2 showed the highest percentage of immature neural-like cells (β-tubulin III⁺), but the neural-like state was transient and reversible. Protocol 3 caused ADSCs to differentiate spontaneously into immature neural-like cells, but not into mature neural cell types. The neural-like cells produced by Protocol 1 lived the longest in culture with little cell death, but Protocol 2 and 3 led to the significant cell death. Therefore, Protocol 1 is the most efficient among these protocols. Additionally, soon after differentiation, the mRNA levels of nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) in dADSCs were sharply decreased by Protocol 1 and 2 (acute induction protocol), but not by Protocol 3 (chronic induction protocol). The results indicate that NTFs played an important role in neural differentiation via acute responses to NGF and BDNF, but not chronically during the transdifferentiation process.     

Transplantation of Mesenchymal Cells Improves Peripheral Limb Ischemia in Diabetic Rats

Adipose tissue-derived mesenchymal stromal cells (ADSCs) are a prominent cellular source for regenerative medicine. We tested whether transplantation of ADSCs into the ischemic muscular tissue of diabetic animals would attenuate impaired cell metabolism and microcirculatory function. We induced unilateral hind limb ischemia in male streptozotocin-treated rats and nondiabetic controls. One day after femoral artery ligation, six rats per group were intramuscularly injected allogeneic ADSCs (10⁶–10⁷–10⁸ cells/mL); or conditioned media from ADSC cultures (CM); or saline; or allogeneic fibroblasts (10⁷ cells/mL); or nonconditioned medium. Rats underwent magnetic resonance angiography; short time inversion recovery (STIR) edema-weighed imaging; proton MR spectroscopy (¹H-MRS); immunoblotting and immunofluorescence on both hind limbs for 4 weeks. T₁-weighted and STIR images showed tissue swelling and signal hyperintensity, respectively, in the ischemic tissue. The mean total ratio of creatine/water for the occluded limbs was significantly lower than for the nonoccluded limbs in both nondiabetic and diabetic rats. ADSC and CM groups had greater recovery of tCr/water in ischemic limbs in both diabetic and nondiabetic rats, with increased expression of α-sarcomeric actinin, vascular endothelial growth factor and hepatocyte growth factor, as well as increased vessel density. ADSCs improve ischemic muscle metabolism and increase neovasculogenesis in diabetic rats.     

Impact of IFN-β and LIF overexpression on human adipose-derived stem cells properties

Adipose-derived stem cells (ADSCs) are a subset of mesenchymal stem cells that their therapeutic effects in various diseases make them an interesting tool in cell therapy. In the current study, we aimed to overexpress interferon-β (IFN-β) and leukemia inhibitory factor (LIF) cytokines in human ADSCs to evaluate the impact of this overexpression on human ADSCs properties. Here, we designed a construct containing IFN-β and LIF and then, transduced human adipose-derived stem cells (hADSCs) by this construct via a lentiviral vector (PCDH-513B). We assessed the ability of long-term expression of the transgene in transduced cells by western blot analysis and enzyme-linked immunosorbent assay techniques on Days 15, 45, and 75 after transduction. For the evaluation of stem cell properties, flow cytometry and differentiation assays were performed. Finally, the MTT assay was done to assess the proliferation of transduced cells compares to controls. Our results showed high-efficiency transduction with highest expression rates on Day 75 after transduction which were 70 pg/ml for IFN-β and 77.9 pg/ml for LIF in comparison with 25.60 pg/ml and 27.63 pg/ml, respectively, in untransduced cells (p = .0001). Also, transduced cells expressed a high level of ADSCs surface markers and successfully differentiated into adipocytes, chondrocytes, neural cells, and osteocytes besides the preservation rate of proliferation near untreated cells (p = .88). All in all, we successfully constructed an hADSC population stably overexpressed IFN-β and LIF cytokines. Considering the IFN-β and LIF anti-inflammatory and neuroprotective effects as well as immune-regulatory properties of hADSCs, the obtained cells of this study could be subjected for further evaluations in experimental autoimmune encephalomyelitis mice model.     

Effect of a subset of adipose-derived stem cells isolated with liposome magnetic beads to promote cartilage repair

This study aimed to investigate the ability of CD146⁺ subset of ADSCs to repair cartilage defects. In this study, we prepared CD146⁺ liposome magnetic beads (CD146⁺LMB) to isolate CD146⁺ADSCs. The cells were induced for chondrogenic differentiation and verified by cartilage-specific mRNA and protein expression. Then a mouse model of cartilage defect was constructed and treated by filling the induced cartilage cells into the damaged joint, to evaluate the function of such cells in the cartilage microenvironment. Our results demonstrated that the CD146⁺LMBs we prepared were uniform, small and highly stable, and cell experiments showed that the CD146⁺LMB has low cytotoxicity to the ADSCs. ADSCs isolated with CD146⁺LMB were all CD146⁺, CD105⁺, CD166⁺ and CD73⁺. After chondrogenic induction, the cells showed significantly increased expression of cartilage markers Sox9, collagen Ⅱ and aggrecan at protein level and significantly increased Sox9, collagen Ⅱ and aggrecan at mRNA level, and the protein expression and mRNA expression of CD146⁺ADSCs group were higher than those of ADSCs group. The CD146⁺ADSCs group showed superior tissue repair ability than the ADSCs group and blank control group in the animal experiment, as judged by gross observation, histological observation and histological scoring. The above results proved that CD146⁺LMB can successfully isolate the CD146⁺ADSCs, and after chondrogenic induction, these cells successfully promoted repair of articular cartilage defects, which may be a new direction of tissue engineering.     

Smooth Muscle-Like Cells Generated from Human Mesenchymal Stromal Cells Display Marker Gene Expression and Electrophysiological Competence Comparable to Bladder Smooth Muscle Cells

The use of mesenchymal stromal cells (MSCs) differentiated toward a smooth muscle cell (SMC) phenotype may provide an alternative for investigators interested in regenerating urinary tract organs such as the bladder where autologous smooth muscle cells cannot be used or are unavailable. In this study we measured the effects of good manufacturing practice (GMP)-compliant expansion followed by myogenic differentiation of human MSCs on the expression of a range of contractile (from early to late) myogenic markers in relation to the electrophysiological parameters to assess the functional role of the differentiated MSCs and found that differentiation of MSCs associated with electrophysiological competence comparable to bladder SMCs. Within 1–2 weeks of myogenic differentiation, differentiating MSCs significantly expressed alpha smooth muscle actin (αSMA; ACTA2), transgelin (TAGLN), calponin (CNN1), and smooth muscle myosin heavy chain (SM-MHC; MYH11) according to qRT-PCR and/or immunofluorescence and Western blot. Voltage-gated Na⁺ current levels also increased within the same time period following myogenic differentiation. In contrast to undifferentiated MSCs, differentiated MSCs and bladder SMCs exhibited elevated cytosolic Ca²⁺ transients in response to K⁺-induced depolarization and contracted in response to K⁺ indicating functional maturation of differentiated MSCs. Depolarization was suppressed by Cd²⁺, an inhibitor of voltage-gated Ca²⁺-channels. The expression of Na⁺-channels was pharmacologically identified as the Na_v1.4 subtype, while the K⁺ and Ca²⁺ ion channels were identified by gene expression of KCNMA1, CACNA1C and CACNA1H which encode for the large conductance Ca²⁺-activated K⁺ channel BK_Ca channels, Ca_v1.2 L-type Ca²⁺ channels and Ca_v3.2 T-type Ca²⁺ channels, respectively. This protocol may be used to differentiate adult MSCs into smooth muscle-like cells with an intermediate-to-late SMC contractile phenotype exhibiting voltage-gated ion channel activity comparable to bladder SMCs which may be important for urological regenerative medicine applications.     

Insulin-Producing Cells Differentiated from Human Bone Marrow Mesenchymal Stem Cells In Vitro Ameliorate Streptozotocin-Induced Diabetic Hyperglycemia

Background

The two major obstacles in the successful transplantation of islets for diabetes treatment are inadequate supply of insulin-producing tissue and immune rejection. Induction of the differentiation of human bone marrow-derived mesenchymal stem cells (hMSCs) into insulin-producing cells (IPCs) for autologous transplantation may alleviate those limitations.

Methods

hMSCs were isolated and induced to differentiate into IPCs through a three-stage differentiation protocol in a defined media with high glucose, nicotinamide, and exendin-4. The physiological characteristics and functions of IPCs were then evaluated. Next, about 3 × 10⁶ differentiated cells were transplanted into the renal sub-capsular space of streptozotocin (STZ)-induced diabetic nude mice. Graft survival and function were assessed by immunohistochemistry, TUNEL staining and measurements of blood glucose levels in the mice.

Results

The differentiated IPCs were characterized by Dithizone (DTZ) positive staining, expression of pancreatic β-cell markers, and human insulin secretion in response to glucose stimulation. Moreover, 43% of the IPCs showed L-type Ca²⁺ channel activity and similar changes in intracellular Ca²⁺ in response to glucose stimulation as that seen in pancreatic β-cells in the process of glucose-stimulated insulin secretion. Transplantation of functional IPCs into the renal subcapsular space of STZ-induced diabetic nude mice ameliorated the hyperglycemia. Immunofluorescence staining revealed that transplanted IPCs sustainably expressed insulin, c-peptide, and PDX-1 without apparent apoptosis in vivo.

Conclusions

IPCs derived from hMSCs in vitro can ameliorate STZ-induced diabetic hyperglycemia, which indicates that these hMSCs may be a promising approach to overcome the limitations of islet transplantation.     

In Situ Transplantation of Alginate Bioencapsulated Adipose Tissues Derived Stem Cells (ADSCs) via Hepatic Injection in a Mouse Model

Objective

Adipose tissue derived stem cells (ADSCs) transplantation has recently gained widespread enthusiasm, particularly in the perspective to use them as potential alternative cell sources for hepatocytes in cell based therapy, mainly because of their capability of hepatogenic differentiation in vitro and in vivo. But some challenges remain to be addressed, including whether ADSCs can be provided effectively to the target organ and whether subsequent proliferation of transplanted cells can be achieved. To date, intrasplenic injection is the conventional method to deliver ADSCs into the liver; however, a number of donor cells retained in the spleen has been reported. In this study, our objective is to evaluate a novel route to transplant ADSCs specifically to the liver. We aimed to test the feasibility of in situ transplantation of ADSCs by injecting bioencapsulated ADSCs into the liver in mouse model.

Methods

The ADSCs isolated from human alpha 1 antitrypsin (M-hAAT) transgenic mice were used to allow delivered ADSCs be readily identified in the liver of recipient mice, and alginate was selected as a cell carrier. We first evaluated whether alginate microspheres are implantable into the liver tissue by injection and whether ADSCs could migrate from alginate microspheres (study one). Once proven, we then examined the in vivo fate of ADSCs loaded microspheres in the liver. Specifically, we evaluated whether transplanted, undifferentiated ASDCs could be induced by the local microenvironment toward hepatogenic differentiation and the distribution of surviving ADSCs in major tissue organs (study two).

Results

Our results indicated ADSCs loaded alginate microspheres were implantable into the liver. Both degraded and residual alginate microspheres were observed in the liver up to three weeks. The viable ADSCs were detectable surrounding degraded and residual alginate microspheres in the liver and other major organs such as bone marrow and the lungs. Importantly, transplanted ADSCs underwent hepatogenic differentiation to become cells expressing albumin in the liver. These findings improve our understanding of the interplay between ADSCs (donor cells), alginate (biomaterial), and local microenvironment in a hepatectomized mouse model, and might improve the strategy of in situ transplantation of ADSCs in treating liver diseases.     

Priming with oxytocin and relaxin improves cardiac differentiation of adipose tissue–derived stem cells

Previous studies have identified the heart as a source and a target tissue for oxytocin and relaxin hormones. These hormones play important roles in the regulation of cardiovascular function and repair of ischemic heart injury. In the current study, we examined the impact of oxytocin and relaxin on the development of cardiomyocytes from mesenchymal stem cells. For this purpose, mouse adipose tissue–derived stem cells (ADSCs) were treated with different concentrations of oxytocin or relaxin for 4 days. Three weeks after initiation of cardiac induction, differentiated ADSCs expressed cardiac-specific genes, Gata4, Mef2c, Nkx2.5, Tbx5, α- and β-Mhc, Mlc2v, Mlc2a and Anp, and cardiac proteins including connexin 43, desmin and α-actinin. 10 ⁻⁷ M oxytocin and 50 ng/mL relaxin induced the maximum upregulation in the expression of cardiac markers. A combination of oxytocin and relaxin induced cardiomyocyte differentiation more potently than the individual factors. In our experiment, oxytocin-relaxin combination increased the population of cardiac troponin I-expressing cells to 6.84% as compared with 2.36% for the untreated ADSCs, 3.7% for oxytocin treatment and 3.41% for relaxin treatment groups. In summary, the results of this study indicated that oxytocin and relaxin hormones individually and in combination can improve cardiac differentiation of ADSCs, and treatment of the ADSCs and possibly other mesenchymal stem cells with these hormones may enhance their cardiogenic differentiation and survival after transplantation into the ischemic heart tissue.