Recovery Of Mesenchymal Stem Cells Research Articles

Scalable mesenchymal stem cell enrichment from bone marrow aspirate using deterministic lateral displacement (DLD) microfluidic sorting.

The growing interest in regenerative medicine has opened new avenues for novel cell therapies using stem cells. Bone marrow aspirate (BMA) is an important source of stromal mesenchymal stem cells (MSCs). Conventional MSC harvesting from BMA relies on archaic centrifugation methods, often leading to poor yield due to osmotic stress, high centrifugation force, convoluted workflow, and long experimental time (∼2-3 hours). To address these issues, we have developed a scalable microfluidic technology based on deterministic lateral displacement (DLD) for MSC isolation. This passive, label-free cell sorting method capitalizes on the morphological differences between MSCs and blood cells (platelets and RBCs) for effective separation using an inverted L-shaped pillar array. To improve throughput, we developed a novel multi-chip DLD system that can process 2.5 mL of raw BMA in 20 ± 5 minutes, achieving a 2-fold increase in MSC recovery compared to centrifugation methods. Taken together, we envision that the developed DLD platform will enable fast and efficient isolation of MSCs from BMA for effective downstream cell therapy in clinical settings.

Fluidic Device System for Mechanical Processing and Filtering of Human Lipoaspirate Enhances Recovery of Mesenchymal Stem Cells.

Adipose tissue is an easily accessible source of stem and progenitor cells that offers exciting promise as an injectable autologous therapeutic for regenerative applications. Mechanical processing is preferred over enzymatic digestion, and the most common method involves shuffling lipoaspirate between syringes and filtering to produce nanofat. Although nanofat has shown exciting clinical results, the authors hypothesized that new device designs could enhance recovery of stem/progenitor cells through optimization of fluid dynamics principles, integration, and automation. The authors designed and fabricated the emulsification and micronization device (EMD) and the filtration device (FD) to replace the manual nanofat procedures. Using human lipoaspirate samples, the EMD and the FD were optimized and compared to traditional nanofat using ex vivo measurements of cell number, viability, and percentage of mesenchymal stem cells and endothelial progenitor cells. The EMD produced results statistically similar to nanofat, and these findings were confirmed for a cohort of diabetic patients. Combining the FD with the EMD was superior to manually filtered nanofat in terms of both recovered cell percentages (>1.5-fold) and numbers (two- to three-fold). Differences were statistically significant for total mesenchymal stem cells and a DPP4 + /CD55 + subpopulation linked to improved wound healing in diabetes. The new EMD and the FD improved mechanical processing of human lipoaspirate in terms of mesenchymal stem cell enrichment and number compared to traditional nanofat. Future work will seek to investigate the wound healing response both in vitro and in vivo, and to refine the technology for automated operation within clinical settings. The new devices improved mechanical processing of human lipoaspirate in terms of stem cell enrichment and number compared to traditional methods. Future work will seek to validate wound healing response and refine the technology for automated operation within clinical settings.

Photothermal release and recovery of mesenchymal stem cells from substrates functionalized with gold nanorods

Mesenchymal stem cell therapies show great promise in regenerative medicine. However, to generate clinically relevant numbers of these stem cells, significant in vitro expansion of the cells is required before transplantation into the affected wound or defect. The current gold standard protocol for recovering in vitro cultured cells involves treatment with enzymes such as trypsin which can affect the cell phenotype and ability to interact with the environment. Alternative enzyme free methods of adherent cell recovery have been investigated, but none match the convenience and performance of enzymatic detachment. In this work we have developed a synthetically simple, low cost cell culture substrate functionalized with gold nanorods that can support cell proliferation and detachment. When these nanorods are irradiated with biocompatible low intensity near infrared radiation (785 nm, 560 mWcm−2) they generate localized surface plasmon resonance induced nanoscale heating effects which trigger detachment of adherent mesenchymal stem cells. Through simulations and thermometry experiments we show that this localized heating is concentrated at the cell-nanorod interface, and that the stem cells detached using this technique show either similar or improved multipotency, viability and ability to differentiate into clinically desirable osteo and adipocytes, compared to enzymatically harvested cells. This proof-of-principle work shows that photothermally mediated cell detachment is a promising method for recovering mesenchymal stem cells from in vitro culture substrates, and paves the way for further studies to scale up this process and facilitate its clinical translation. Statement of significanceNew non-enzymatic methods of harvesting adherent cells without damaging or killing them are highly desirable in fields such as regenerative medicine. Here, we present a synthetically simple, non-toxic, infra-red induced method of harvesting mesenchymal stem cells from gold nanorod functionalized substrates. The detached cells retain their ability to differentiate into therapeutically valuable osteo and adipocytes. This work represents a significant improvement on similar cell harvesting studies due to: its simplicity; the use of clinically valuable stem cells as oppose to immortalized cell lines; and the extensive cellular characterization performed. Understanding, not just if cells live or die but how they proliferate and differentiate after photothermal detachment will be essential for the translation of this and similar techniques into commercial devices.

The morphology and application of stem cells in digestive system surgery.

Stem cells constitute a group of cells which possess the ability to self-renew as well as the capacity to differentiate into a vast number of different cells within the human organism. Moreover, stem cells are able to undergo a potentially unlimited number of divisions and this characteristic is clinically essential. Specific fields of its application include treatment of diseases mainly in the field of haematology, orthopaedics, surgery, dentistry, and neurology. In the following work, the current knowledge concerning mechanisms of stem cell treatment in different parts of the digestive system with its diseases as well as adjacent therapy for surgery has been revised. Stem cells therapy may be used in the treatment of various diseases of different parts of the digestive system. This also applies to the end part of the digestive tract (proctological diseases) because stem cells can be used to treat fistulas. Liposuction allows more recovery of mesenchymal stem cells, compared to previous bone marrow harvesting methods. Despite the application of stem cells in the treatment of different diseases used for many years so far, the therapeutic use for the regeneration of the gastrointestinal tract is still rare and unfamiliar. Regenerative medicine seems to be a promising tool in medical research, especially when insulated cells and designed biomaterials are taken into consideration. Major points of discussion include types of stem cells, their origin or differentiation for the treatment of many diseases.

Cellular Proliferation of Equine Bone Marrow- and Adipose Tissue-Derived Mesenchymal Stem Cells Decline With Increasing Donor Age.

Background: Bone marrow (BM)- and adipose tissue (AT)-derived mesenchymal stem cells (MSCs) are used increasingly for autologous cell therapy in equine practice to treat musculoskeletal and other injuries. Current recommendations often call for 10–100 million MSCs per treatment, necessitating the expansion of primary cells in culture prior to therapeutic use. Of concern, human and rodent studies have shown a decline of both MSC recovery from sampled tissue and in vitro proliferative capacity with increasing donor age. This may be problematic for applications of autologous cell-based therapies in the important equine demographic of older patients.Objectives: To investigate the effect of donor age on the cellular proliferation of equine BM- and AT-MSCs.Study Design: In vitro study.Methods: BM- and AT-MSCs and dermal fibroblasts (biological control) were harvested from horses in five different age groups (n = 4, N = 60); newborn (0 days), yearling (15–17 months), adult (5–8 years), middle-aged (12–18 years), and geriatric (≥22 years). Proliferation of the cells was tested using an EdU incorporation assay and steady state mRNA levels measured for targeted proliferation, aging, and senescence biomarkers.Results: The cellular proliferation of equine BM- and AT-MSCs declined significantly in the geriatric cohort relative to the younger age groups. Proliferation levels in the two MSC types were equally affected by donor age. Analysis of steady state mRNA levels showed an up-regulation in tumor suppressors, apoptotic genes, and multiple growth factors in MSCs from old horses, and a down-regulation of some pro-cycling genes with a few differences between cell types.Main Limitations: Potential age-dependent differences in cell function parameters relevant to cell-therapy application were not investigated.Conclusions: The cellular proliferation of equine BM- and AT-MSCs declined at advanced donor ages. High levels of in vitro proliferation were observed in both MSC types from horses in the age groups below 18 years of age.

Comparison of viability and proliferation potency of stromal vascular fraction stored at different temperature

Background & Aim Adipose tissue provides an abundant source of stromal vascular fraction (SVF), which resides a potential regenerative medicine named mesenchymal stem cells (MSC). Storage period and temperature before isolation may impact the quality of isolated cells. Here, we identify the effect of storage periods and temperatures to SVF viability and cell number along with the MSC recovery. Methods, Results & Conclusion A total of 70 ml donor-adipose tissue was divided into two different groups, room temperature (RT) and 4-8°C. SVF was isolated using manual method. Each group was process after stored for 4, 5, 6, 7, and 12 days. After it passed each incubation time, adipose samples were processed enzymatically until SVF was obtained. The viability of SVF stored at RT was decreasing until 40%, but relatively constant when stored at 4-8°C (>75%). Nevertheless, the sample stored at RT revealed to have better proliferation and colonization than sample stored at 4-8°C. The sample retained the proliferation capability when stored at RT up to 7 days, but only up to 5 days when stored at 4-8°C. The average of SVF number was higher in the sample stored at RT (8,552,315 cell/mL) compared to the sample at 4-8°C (4,581,111 cell/mL). The highest SVF number was observed in sample stored at 4 days before isolation. The average of MSC recovery was also higher at RT compared to sample stored at 4-8°C, which was 6% and 3%, respectively. In conclusion, adipose tissue is preferable to be stored at RT before isolation for higher SVF total number and MSC recovery. The adipose tissue should be processed immadiately or stored only up to 4 days after sample collection.

Combinations of Osmolytes, Including Monosaccharides, Disaccharides, and Sugar Alcohols Act in Concert During Cryopreservation to Improve Mesenchymal Stromal Cell Survival.

There is demand for non-dimethyl sulfoxide (DMSO) cryoprotective agents that maintain cell viability without causing poor postthaw function or systemic toxicity. The focus of this investigation involves expanding our understanding of multicomponent osmolyte solutions and their ability to preserve cell viability during freezing. Controlled cooling rate freezing, Raman microscopy, and differential scanning calorimetry (DSC) were utilized to evaluate the differences in recovery and ice crystal formation behavior for solutions containing multiple cryoprotectants, including sugars, sugar alcohols, and small molecule additives. Postthaw recovery of mesenchymal stem cells (MSCs) in solutions containing multiple osmolytes have been shown to be comparable or better than that of MSCs frozen in 10% DMSO at 1°C/min when the solution composition is optimized. Maximum postthaw recovery was observed in these multiple osmolyte solutions with incubation times of up to 2 h before freezing. Raman images demonstrate large ice crystal formation in cryopreserved cells incubated for shorter periods of time (∼30 min), suggesting that longer permeation times are needed for these solutions. Recovery was dependent upon the concentration of each component in solution, and was not strongly correlated with osmolarity. It is noteworthy that the postthaw recovery varied significantly with the composition of solutions containing the same three components and this variation exhibited an inverted U-shape behavior, indicating that there may be a "sweet spot" for different combinations of osmolytes. Raman images of freezing behavior in different solution compositions were consistent with the observed postthaw recovery. Phase change behavior (solidification patterns and glass-forming tendency) did not differ for solutions with similar osmolarity, but differences in postthaw recovery suggest that biological, not physical, methods of protection are at play. Lastly, molecular substitution of glucose (a monosaccharide) for sucrose (a disaccharide) resulted in a significant drop in recovery. Taken together, the information from these studies increases our understanding of non-DMSO multicomponent cryoprotective solutions and the manner by which they enhance postthaw recovery.

Comparative Characterization of Canine and Human Mesenchymal Stem Cells Derived from Bone Marrow

Bone marrow is a major source of mesenchymal stem cells (MSC), which are used in tissue engineering and other autologous stem cell therapies. Studies designed to use large canine models and translate the results to human practice must take into account the ex vivo and in vitro differences in the bone marrow samples. A set of morphological and physiological markers was used to compare MSC derived from canine and human bone marrow. Despite anticlotting treatment, frequent bone marrow clotting was a problem with canine samples, so we developed a protocol for enzymatic digestion of undesirable clots. We compared colony forming units (CFU) assay, population doubling time (PDT), senescenceassociated β-galactosidase (SA-β-Gal) activity, as well as the ability of cells to differentiate to osteogenic, adipogenic and chondrogenic phenotypes. Urokinase digestion resulted in recovery of MSC: 4-fold more CFU from canine and 1.6-fold more from human samples when compared to untreated samples. Canine MSC were less robust in vitro – they divided actively only for four weeks in culture, while human cells divided for longer than eight weeks. After six weeks in culture, canine MSC underwent 17 population doublings of while human cells reached 26. The percentage of senescent cells increased linearly with time, but with a faster rate in canine MSC. Human and canine MSC underwent differentiation to all lineages; however canine MSC had generally lower differentiation potential. In conclusion, the discrepancy between canine and human cultures must be considered in future MSC-based therapies based on dogs as animal model.

Survivin Is a Master Regulator of Mesenchymal Stem Cell Functions and Potential Therapeutic Target for Mesenchymal Stem Cell Expansion

Mesenchymal stemcells (MSCs) are found throughout adult organisms and are involved in tissue maintenance and repair, as well as in the regulation of hematopoiesis and immunologic responses. Several clinical trials are currently under way to use allogeneic MSCs for enhancement of hematopoietic stem cell transplantation and treatment of graft-versus-host disease, spinal cord injury, cartilage and meniscus repair, and stroke. However, the therapeutic uses of MSCs can be limited by insufficient MSC number, their survival, and their ability to differentiate into multiple lineages, pointing to an important need to identify factors regulating MSC survival, proliferation, and differentiation. In this study, we explored the role of the anti-apototic protein Survivin in MSC functions. Survivin is an intracellular member of the inhibitor-of-apoptosis protein (IAP) family and is implicated in regulation of apoptosis, cell division, and cell cycle control of many cell types including hematopoietic stem cells, leukemic cells, and endothelial cells. However, whether Survivin is involved in MSC activity or function is unknown. Using flow cytometry and function assays, we found that adult mouse and human MSCs, identified as CD45-Ter119-CD51+PDGFR+Nestin+ cells, express high levels of Survivin, which regulates MSCs survival and expansion under normal physiological and stressful conditions. Flow cytometry analysis revealed that approximately 40.5±3.6% of mouse bone marrow MSCs, 33.6±4.2% of adult human bone marrow MSCs, and 43.6±5.3% human cord blood MSCs express Survivin. Treatment of mice in vivo with YM155 (10 mg/kg), a small molecule Survivin inhibitor, for 6 days decreased bone marrow CD45-Ter119-CD51+PDGFR+Nestin+ MSC by 2.1 fold and functional fibroblast colony formation (CFU-F) by 2.8 fold. Survivin gene deletion using Survivin-specific SiRNA decreased mouse and human bone marrow derived MSCs number and CFU-F ability by 3.2 fold and 2.8 fold respectively. Retroviral overexpression of Survivin in mouse MSCs enhanced CFU-F formation by 4 fold. In an in vitro wound healing assay YM155-treated MSCs recovered more slowly compared to control cultures. To determine underlying mechanisms involved in Survivin dependent regulation of MSC function, we measured the survival and proliferation of YM155 treated MSCs. Treatment of mouse or human bone marrow-derived MSCs with YM155 (50 nmol/l) for 48 hours enhanced the caspase 3 and 7 expression by 42.8% and 63.9% respectively, while BrdU incorporation was similar in control and YM155-treated MSCs, suggesting that Survivin is primarily involved in MSC survival. To explore whether Survivin is also involved in growth factor-mediated MSC survival and expansion, mouse bone marrow derived MSCs were cultured in the presence of platelet derived growth factor (PDGF) or basic fibroblast growth factor (b-FGF) with or without the Survivin inhibitor YM155. PDGF enhanced the Survivin expression by 52.8±2.2% and b-FGF enhanced Survivin expression by 43.8±4.5% and increased the CFU-F counts by 5.6 fold and 4.2 fold, respectively. In contrast, YM155 treated MSCs did not show any enhancement in Survivin expression, and CFU-F were substantially lower than the corresponding controls (PDGF or b-FGF treated cultures). To determine whether Survivin affects the Survival of stressed MSCs, we exposed mouse bone marrow MSCs to 4 Gy irradiation dose and followed by culture for 3 days with or without the Survivin inhibitor YM155. Irradiation alone reduced the survival of MSCs by 53.5%, however the viability of irradiated MSCs treated with YM155 was reduced by 82.8%. The hallmark of MSC function is the capacity to differentiate into several mesodermal lineages. We found that the exposure of MSC cultures to YM155 during the lineage differentiation process inhibits MSC osteogenic and adipogenic differentiation while sparing chondrogenic cell differentiation. In conclusion, our study demonstrates that Survivin controls basal and growth factor dependent survival and expansion of mouse and human MSCs, and protects them from irradiation induced cell death. Furthermore, our data suggest the regulating Survivin expression in MSCs would be beneficial to enhance MSC recovery for clinical utility. Disclosures No relevant conflicts of interest to declare.

Freezing and recovery of mesenchymal stem cells in human platelet lysate

Freezing and recovery of mesenchymal stem cells in human platelet lysate

Effectiveness of human mesenchymal stem cells derived from bone marrow cryopreserved for 23–25 years

ObjectiveTo evaluate long-term cryopreserved human bone marrow cells (BMCs) as a source of functional mesenchymal stem cells (MSCs). MethodsSamples of human BMCs that were cryopreserved for 23–25years (n=20) were thawed to obtain an initial culture and a primary culture (P0) that was propagated through five passages (P1–P5) to obtain MSCs. Freshly collected human bone marrow samples (n=20) were used as controls for comparison of efficiency of recovery and growth characteristics of MSCs. P3 cultures were tested for their capacity to differentiate into osteoblasts, adipocytes, and neuronal cells. Appropriate staining, immunohistochemical and biochemical methods were employed to ascertain cell type identities at different stages of culturing. ResultsIn the initial culture, the cell adherence rate of the cryopreserved cells was significantly lower than that of controls (19.7% vs. 38.2%, p<0.05) while the relative rate of recovery of MSCs was only 48.5±8.6% in P0. At the end of P3, fibroblast-like cells accounted for about 95% of cells in both cryopreserved and control groups (p>0.05). These cells were positive for essential MSC surface molecules (CD90, CD105, CD166, CD44, CD29, CD71, CD73) and negative for haematopoietic and endothelial cell markers (CD45, CD34, HLA-DR). The cell growth and cell cycle patterns were similar for both groups. MSCs at P3 from both groups had similar capacities to differentiate in vitro into osteoblasts, adipocytes, and neuronal cells. ConclusionUsing the methods described here, long-term (23–25years) cryopreserved human BMCs can be successfully cultivated to obtain MSCs that have good differentiation capabilities.

Isolation of the Stromal/Vascular Fraction of Murine Bone Marrow Dramatically Enhances MSC Yield, Allows Isolation of Marrow Endothelial Cells and Reveals Multiple Subpopulations of Stromal Cells.

AbstractAbstract 3861The bone marrow is the organ of residence of a population of multipotent progenitor cells most commonly referred to as mesenchymal stem cells (MSC) based upon their multilineage differentiation potential into bone, cartilage and adipose tissue. The capacity for MSC to contribute to tissue repair demonstrated by numerous previous reports has engendered considerable interest in their application to a broad range of cellular therapies. It follows that a robust reproducible methodology for obtaining high yields of MSC from preclinical animal models, such as rodents, would greatly facilitate the development of these various MSC-based cellular therapies. The plastic-adherent, clonogenic progenitors termed colony forming unit-fibroblast (CFU-F) originally identified by Freidenstein and colleagues that initiate MSC cultures are a rare population in the marrow of all mammalian species so far examined. This is particularly so in the case of the mouse where reported incidences of CFU-F are typically in the range of 1/200,000 bone marrow BM cells. The low incidence of CFU-F significantly complicates the isolation of homogeneous populations of MSC from mouse BM, a common problem being contamination with hematopoietic cells. Seeking to develop an improved methodology to harvest MSC from mouse using methods based on plastic adherent bone marrow, we took advantage of burgeoning evidence demonstrating the perivascular location of MSC not only in the bone marrow, but also in multiple tissues. We hypothesized that a potential reason for the low yield of mMSC from mBM is the flushing of the marrow used to remove single cells suspensions and the consequent destruction of the marrow vasculature, which may adversely affect recovery of MSC physically associated with the abluminal surface of blood vessels. Herein, we describe a simple methodology based on preparation of intact marrow plugs that yields distinct populations of both stromal and endothelial cells. BM plugs are subjected to 3 sequential rounds of digestion in collagenase/dispase and each fraction assayed for content of CFU-F. The recovery of CFU-F obtained by pooling the product of each digestion (1643+199) reproducibly exceeds that obtained using the standard BM flushing technique (13.3+1.9) by at least 2 orders of magnitude (P=<0.001; N = 8) with an accompanying 196-fold enrichment of CFU-F frequency. Purified BM stromal cell populations devoid of hematopoietic contamination are readily obtained by FACS at P0 and these demonstrate robust multilineage differentiation into bone, adipose and chondrogenic progeny using standard in vitro bioassays. A detailed immunophenotypic analysis of the P0 cultures demonstrated the existence of multiple stromal cell subpopulations many of the markers analyzed, including Sca-1, CD90, CD105, CD146 and PDGFRa, which was progressively lost with serial passaging. Discrete subpopulations of stromal cells identified at P0, in many cases had phenotypically identical counterparts in the BM cell suspensions prepared by serial digestion and we are in the process of quantitatively analyzing the evolution of selected phenotypes in vitro to provide clues as to the identity of the founder population of stromal cells that gives rise to ‘MSC’ in vitro. Finally, the phenotypic analysis of P0 cultures also revealed a discrete population of CD105BrightPDGFRaNeg cells representing a mean of 26.7% of hematopoietic lineage-negative cells. Upon isolation and serial propagation, the cells maintain expression of all of the vascular endothelial markers examined including CD31, CD105, VCAM-1, CD144 and MECA32 and also demonstrate inducible expression of E-selectin upon treatment with TNF-a. In conclusion, we describe a simple and robust methodology that, for the first time, allows the simultaneous isolation of both the stromal and vascular components of mouse BM. Secondly, the yield of ‘MSC’ afforded by this technique far exceeds that reported in any previous study. Thirdly, this technique reveals a level of stromal cell heterogeneity not apparent in previous analyses of mouse BM-derived MSC that more realistically reflects the likely complexity of stromal cell populations in vivo and represents a platform for the eventual prospective isolation of specific subpopulations. These studies will greatly enhance experimental strategies designed to analyze not only MSC identity but also the function of the vascular hematopoietic niche. Disclosures:No relevant conflicts of interest to declare.

Optimisation of bone marrow aspiration from the equine sternum for the safe recovery of mesenchymal stem cells

Mesenchymal stem cell (MSC) therapy for orthopaedic disease is being used with increasing frequency; there is a need to define a safe, reliable and effective technique for the recovery of MSCs from the sternum of the horse. To describe an optimised safe technique for obtaining bone marrow-derived MSCs from the sternum of the Thoroughbred horse. The anatomical relationship of the sternum with the heart and internal anatomy was demonstrated in cadavers. Sternal anatomy was evaluated ultrasonographically and after midline sectioning. Sternebrae were examined histologically after aspiration to determine the effect of needle insertion. The quality of the aspirate was evaluated as the number of colony-forming units from sequential and separately aspirated 5 ml aliquots and assessed for their multipotency using trilineage differentiation. The optimal safe location for the needle was the 5th sternebra because it had a safe dorsoventral thickness and was cranial to the apex of the heart. This sternebra could be reliably identified ultrasonographically. Aspirates could also be obtained from the 4th and 6th sternebrae, although the former is between the front limbs and the latter closer to the heart. Minimal disruption of the internal bony architecture was seen after needle insertion through the thin outer cortex and the first 5 ml aliquot contained the greatest number of colony-forming units of mesenchymal stem cells with trilineage capabilities. Accurate placement of a Jamshidi needle into the medullary cavity of the 4th-6th individual sternebrae is facilitated by the use of ultrasonography and enables aspiration of bone marrow reliably with minimal damage to the sternum and risk to the horse. Sternal marrow aspiration as described is a safe and reliable technique to obtain MSCs for orthopaedic cell-based therapies.

Term amniotic membrane is a high throughput source for multipotent mesenchymal stem cells with the ability to differentiate into endothelial cells in vitro

BackgroundTerm Amniotic membrane (AM) is a very attractive source of Mesenchymal Stem Cells (MSCs) due to the fact that this fetal tissue is usually discarded without ethical conflicts, leading to high efficiency in MSC recovery with no intrusive procedures. Here we confirmed that term AM, as previously reported in the literature, is an abundant source of hMSCs; in particular we further investigated the AM differentiation potential by assessing whether these cells may also be committed to the angiogenic fate. In agreement with the recommendation of the International Society for Cellular Therapy, the mesenchymal cells herein investigated were named Amniotic Membrane-human Mesenchymal Stromal Cells (AM-hMSC).ResultsThe recovery of hMSCs and their in vitro expansion potential were greater in amniotic membrane than in bone marrow stroma. At flow cytometry analysis AM-hMSCs showed an immunophenotypical profile, i.e., positive for CD105, CD73, CD29, CD44, CD166 and negative for CD14, CD34, CD45, consistent with that reported for bone marrow-derived MSCs. In addition, amniotic membrane-isolated cells underwent in vitro osteogenic (von Kossa stain), adipogenic (Oil Red-O stain), chondrogenic (collagen type II immunohistochemichal detection) and myogenic (RT-PCR MyoD and Myogenin expression as well as desmin immunohistochemical detection) differentiation. In angiogenic experiments, a spontaneous differentiation into endothelial cells was detected by in vitro matrigel assay and this behaviour has been enhanced through Vascular Endothelial Growth Factor (VEGF) induction. According to these findings, VEGF receptor 1 and 2 (FLT-1 and KDR) were basally expressed in AM-hMSCs and the expression of endothelial-specific markers like FLT-1 KDR, ICAM-1 increased after exposure to VEGF together with the occurrence of CD34 and von Willebrand Factor positive cells.ConclusionThe current study suggests that AM-hMSCs may emerge as a remarkable tool for the cell therapy of multiple diseased tissues. AM-hMSCs may potentially assist both bone and cartilage repair, nevertheless, due to their angiogenic potential, they may also pave the way for novel approaches in the development of tissue-engineered vascular grafts which are useful when vascularization of ischemic tissues is required.

Interleukin-17A: A T-Cell-Derived Growth Factor for Murine and Human Mesenchymal Stem Cells

Interleukin-17A (IL-17A) is a proinflammatory cytokine expressed in activated T-cells. It is required for microbial host defense and is a potent stimulator of granulopoiesis. In a dose-dependent fashion, IL-17A expanded human mesenchymal stem cells (MSCs) and induced the proliferation of mature stroma cells in bone marrow-derived stroma cultures. Recombinant human interleukin-17A (rhIL-17A) nearly doubled colony-forming unit-fibroblast (CFU-f) frequency and almost tripled the surface area covered by stroma. In a murine transplant model, in vivo murine (m)IL-17A expression enhanced CFU-f by 2.5-fold. Enrichment of the graft with CD4(+) T-cell resulted in a 7.5-fold increase in CFU-f in normal C57BL/6, but only threefold in IL-17Ra(-/-) mice on day 14 post-transplant. In this transplant model, in vivo blockade of IL-17A in C57BL/6 mice resembled the phenotype of IL-17Ra(-/-) mice. Approximately half of the T-cell-mediated effect on MSC recovery following radiation-conditioned transplantation was attributed to the IL-17A/IL-17Ra pathway. Pluripotent MSCs have the potential of regenerating various tissues, and mature stroma cells are critical elements of the hematopoietic microenvironment (HME). The HME is pivotal for formation and maintenance of functional blood cells. As a newly identified stroma cell growth factor, IL-17A might have potential applications for novel treatment approaches involving MSCs, such as tissue graft engineering.