Microfluidic single-cell transcriptional analysis rationally identifies novel surface marker profiles to enhance cell-based therapies
Finding a needle in a "needlestack".
Cell cycle (Georgetown, Tex.)
Progenitor Cell Dysfunctions Underlie Some Diabetic Complications
AMERICAN JOURNAL OF PATHOLOGY
2015; 185 (10): 2607-2618
Transdermal deferoxamine prevents pressure-induced diabetic ulcers.
Proceedings of the National Academy of Sciences of the United States of America
2015; 112 (1): 94-99
Current progenitor cell therapies have only modest efficacy, which has limited their clinical adoption. This may be the result of a cellular heterogeneity that decreases the number of functional progenitors delivered to diseased tissue, and prevents correction of underlying pathologic cell population disruptions. Here, we develop a high-resolution method of identifying phenotypically distinct progenitor cell subpopulations via single-cell transcriptional analysis and advanced bioinformatics. When combined with high-throughput cell surface marker screening, this approach facilitates the rational selection of surface markers for prospective isolation of cell subpopulations with desired transcriptional profiles. We establish the usefulness of this platform in costly and highly morbid diabetic wounds by identifying a subpopulation of progenitor cells that is dysfunctional in the diabetic state, and normalizes diabetic wound healing rates following allogeneic application. We believe this work presents a logical framework for the development of targeted cell therapies that can be customized to any clinical application.
View details for DOI 10.1038/ncomms11945
View details for Web of Science ID 000379085200001
View details for PubMedID 27324848
Tracking the elusive fibrocyte: identification and characterization of collagen-producing hematopoietic lineage cells during murine wound healing.
2014; 32 (5): 1347-1360
There is a high mortality in patients with diabetes and severe pressure ulcers. For example, chronic pressure sores of the heels often lead to limb loss in diabetic patients. A major factor underlying this is reduced neovascularization caused by impaired activity of the transcription factor hypoxia inducible factor-1 alpha (HIF-1α). In diabetes, HIF-1α function is compromised by a high glucose-induced and reactive oxygen species-mediated modification of its coactivator p300, leading to impaired HIF-1α transactivation. We examined whether local enhancement of HIF-1α activity would improve diabetic wound healing and minimize the severity of diabetic ulcers. To improve HIF-1α activity we designed a transdermal drug delivery system (TDDS) containing the FDA-approved small molecule deferoxamine (DFO), an iron chelator that increases HIF-1α transactivation in diabetes by preventing iron-catalyzed reactive oxygen stress. Applying this TDDS to a pressure-induced ulcer model in diabetic mice, we found that transdermal delivery of DFO significantly improved wound healing. Unexpectedly, prophylactic application of this transdermal delivery system also prevented diabetic ulcer formation. DFO-treated wounds demonstrated increased collagen density, improved neovascularization, and reduction of free radical formation, leading to decreased cell death. These findings suggest that transdermal delivery of DFO provides a targeted means to both prevent ulcer formation and accelerate diabetic wound healing with the potential for rapid clinical translation.
View details for DOI 10.1073/pnas.1413445112
View details for PubMedID 25535360
Surface Tethered Epidermal Growth Factor Protects Proliferating and Differentiating Multipotential Stromal Cells from FasL-Induced Apoptosis
2013; 31 (1): 104-116
Fibrocytes are a unique population of circulating cells reported to exhibit characteristics of both hematopoietic and mesenchymal cells, and play an important role in wound healing. However, putative fibrocytes have been found to lose expression of hematopoietic surface markers such as CD45 during differentiation, making it difficult to track these cells in vivo with conventional methodologies. In this study, to distinguish hematopoietic and nonhematopoietic cells without surface markers, we took advantage of the gene vav 1, which is expressed solely on hematopoietic cells but not on other cell types, and established a novel transgenic mouse, in which hematopoietic cells are irreversibly labeled with green fluorescent protein and nonhematopoietic cells with red fluorescent protein. Use of single-cell transcriptional analysis in this mouse model revealed two discrete types of collagen I (Col I) expressing cells of hematopoietic lineage recruited into excisional skin wounds. We confirmed this finding on a protein level, with one subset of these Col I synthesizing cells being CD45+ and CD11b+, consistent with the traditional definition of a fibrocyte, while another was CD45- and Cd11b-, representing a previously unidentified population. Both cell types were found to initially peak, then reduce posthealing, consistent with a disappearance from the wound site and not a loss of identifying surface marker expression. Taken together, we have unambiguously identified two cells of hematopoietic origin that are recruited to the wound site and deposit collagen, definitively confirming the existence and natural time course of fibrocytes in cutaneous healing. Stem Cells 2014;32:1347-1360.
View details for DOI 10.1002/stem.1648
View details for PubMedID 24446236
Multiple Subsets of Brain Tumor Initiating Cells Coexist in Glioblastoma
2016; 34 (6): 1702-1707
Multipotential stromal cells or mesenchymal stem cells (MSCs) have been proposed as aids in regenerating bone and adipose tissues, as these cells form osteoblasts and adipocytes. A major obstacle to this use of MSC is the initial loss of cells postimplantation. This cell death in part is due to ubiquitous nonspecific inflammatory cytokines such as FasL generated in the implant site. Our group previously found that soluble epidermal growth factor (sEGF) promotes MSC expansion. Furthermore, tethering EGF (tEGF) onto a two-dimensional surface altered MSC responses, by restricting epidermal growth factor receptor (EGFR) to the cell surface, causing sustained activation of EGFR, and promoting survival from FasL-induced death. sEGF by causing internalization of EGFR does not support MSC survival. However, for tEGF to be useful in bone regeneration, it needs to allow for MSC differentiation into osteoblasts while also protecting emerging osteoblasts from apoptosis. tEGF did not block induced differentiation of MSCs into osteoblasts, or adipocytes, a common default MSC-differentiation pathway. MSC-derived preosteoblasts showed increased Fas levels and became more susceptible to FasL-induced death, which tEGF prevented. Differentiating adipocytes underwent a reduction in Fas expression and became resistant to FasL-induced death, with tEGF having no further survival effect. tEGF protected undifferentiated MSC from combined insults of FasL, serum deprivation, and physiologic hypoxia. Additionally, tEGF was dominant in the face of sEGF to protect MSC from FasL-induced death. Our results suggest that MSCs and differentiating osteoblasts need protective signals to survive in the inflammatory wound milieu and that tEGF can serve this function.
View details for DOI 10.1002/stem.1215
View details for Web of Science ID 000312561000011
View details for PubMedID 22948863
Extracellular superoxide dismutase deficiency impairs wound healing in advanced age by reducing neovascularization and fibroblast function.
2016; 25 (3): 206-211
Brain tumor-initiating cells (BTICs) are self-renewing multipotent cells critical for tumor maintenance and growth. Using single-cell microfluidic profiling, we identified multiple subpopulations of BTICs co-existing in human glioblastoma, characterized by distinct surface marker expression and single-cell molecular profiles relating to distinct bulk tissue molecular subtypes. These data suggest BTIC subpopulation heterogeneity as an underlying source of intra-tumoral bulk tissue molecular heterogeneity, and will support future studies into BTIC subpopulation-specific therapies. This article is protected by copyright. All rights reserved.
View details for DOI 10.1002/stem.2359
View details for Web of Science ID 000378089500025
View details for PubMedID 26991945
Adipose-Derived Stem Cell-Seeded Hydrogels Increase Endogenous Progenitor Cell Recruitment and Neovascularization in Wounds
TISSUE ENGINEERING PART A
2016; 22 (3-4): 295-305
Advanced age is characterized by impairments in wound healing, and evidence is accumulating that this may be due in part to a concomitant increase in oxidative stress. Extended exposure to reactive oxygen species (ROS) is thought to lead to cellular dysfunction and organismal death via the destructive oxidation of intra-cellular proteins, lipids and nucleic acids. Extracellular superoxide dismutase (ecSOD/SOD3) is a prime antioxidant enzyme in the extracellular space that eliminates ROS. Here, we demonstrate that reduced SOD3 levels contribute to healing impairments in aged mice. These impairments include delayed wound closure, reduced neovascularization, impaired fibroblast proliferation and increased neutrophil recruitment. We further establish that SOD3 KO and aged fibroblasts both display reduced production of TGF-β1, leading to decreased differentiation of fibroblasts into myofibroblasts. Taken together, these results suggest that wound healing impairments in ageing are associated with increased levels of ROS, decreased SOD3 expression and impaired extracellular oxidative stress regulation. Our results identify SOD3 as a possible target to correct age-related cellular dysfunction in wound healing.
View details for DOI 10.1111/exd.12909
View details for PubMedID 26663425
Sutureless Microsurgical Anastomosis Using an Optimized Thermoreversible Intravascular Poloxamer Stent.
Plastic and reconstructive surgery
2016; 137 (2): 546-556
Adipose-derived mesenchymal stem cells (ASCs) are appealing for cell-based wound therapies because of their accessibility and ease of harvest, but their utility is limited by poor cell survival within the harsh wound microenvironment. In prior work, our laboratory has demonstrated that seeding ASCs within a soft pullulan-collagen hydrogel enhances ASC survival and improves wound healing. To more fully understand the mechanism of this therapy, we examined whether ASC-seeded hydrogels were able to modulate the recruitment and/or functionality of endogenous progenitor cells. Employing a parabiosis model and fluorescence-activated cell sorting analysis, we demonstrate that application of ASC-seeded hydrogels to wounds, when compared with injected ASCs or a noncell control, increased the recruitment of provascular circulating bone marrow-derived mesenchymal progenitor cells (BM-MPCs). BM-MPCs comprised 23.0% of recruited circulating progenitor cells in wounds treated with ASC-seeded hydrogels versus 8.4% and 2.1% in those treated with controls, p < 0.05. Exploring the potential for functional modulation of BM-MPCs, we demonstrate a statistically significant increase in BM-MPC migration, proliferation, and tubulization when exposed to hydrogel-seeded ASC-conditioned medium versus control ASC-conditioned medium (73.8% vs. 51.4% scratch assay closure; 9.1% vs. 1.4% proliferation rate; 10.2 vs. 5.5 tubules/HPF; p < 0.05 for all assays). BM-MPC expression of genes related to cell stemness and angiogenesis was also significantly increased following exposure to hydrogel-seeded ASC-conditioned medium (p < 0.05). These data suggest that ASC-seeded hydrogels improve both progenitor cell recruitment and functionality to effect greater neovascularization.
View details for DOI 10.1089/ten.tea.2015.0277
View details for Web of Science ID 000369987900012
View details for PubMedID 26871860
View details for PubMedCentralID PMC4779321
Epidermal Growth Factor Tethered to β-Tricalcium Phosphate Bone Scaffolds via a High-Affinity Binding Peptide Enhances Survival of Human Mesenchymal Stem Cells/Multipotent Stromal Cells in an Immune-Competent Parafascial Implantation Assay in Mice.
Stem cells translational medicine
Sutureless microvascular anastomosis has great translational potential to simplify microvascular surgery, shorten operative times, and improve clinical outcomes. The authors developed a transient thermoreversible microvascular stent using a poloxamer to maintain vessel lumen patency before application of commercially available adhesives to seal the anastomosis instead of sutures. Despite technical success, human application necessitates bovine serum albumin removal from existing formulations; rapid poloxamer transition between states; and increased stiffness for reliable, reproducible, and precise microvascular approximation.Two commercially available poloxamers were used in this study (P407 and P188). After removing bovine serum albumin, each poloxamer was tested at varying concentrations either alone or in combination to determine the optimal preparation for sutureless microvascular anastomosis. Transition temperature and formulation stiffness were tested in vitro by rheometry, with the most promising combinations tested in an established in vivo model.Increasing poloxamer concentration resulted in an increase in stiffness and decrease in transition temperature. Pure P188 without bovine serum albumin, dissolved in phosphate-buffered saline to a 45% concentration, demonstrated desirable rheologic behavior, with precise gel transition and increased gel stiffness compared with our previous formulation of 17% P407 (96 kPa versus 10 kPa). These characteristics were optimal for microsurgical intravascular use, offering surgical precision and control between liquid and solid states, depending on the surgically controlled local temperature.Use of 45% P188 without bovine serum albumin demonstrated optimal rheologic and translational properties as a microvascular stent for sutureless anastomosis. Rapid transition, increased stiffness, and safety profile demonstrate safe translational application for human clinical trials.
View details for DOI 10.1097/01.prs.0000475774.37267.3f
View details for PubMedID 26818289
Improved transplanted stem cell survival in a polymer gel supplemented with tenascin-C accelerates healing and reduces scarring of murine skin wounds.
: Mesenchymal stem cells/multipotent stromal cells (MSCs) are attractive candidates for cell therapies owing to their ability to differentiate into many lineages. However, these cells often fail to survive when implanted into a harsh wound environment, limiting efficacy in vivo. To improve MSC survival, we previously found that tethered epidermal growth factor (tEGF) molecules that restrict epidermal growth factor receptor (EGFR) signaling to the cell surface provide resistance to death signals. To adapt this system to wound healing, we tethered epidermal growth factor (EGF) to tricalcium phosphate (TCP) particle scaffolds, clinically used in bone healing. Human primary MSCs seeded on TCP and mixed into a collagen-based gel were injected in the perifascial space of immunocompetent mice with or without tEGF attached to the surface. We found that tethering EGF to the TCP scaffolds yielded approximately a fourfold increase in MSC survival compared with non-EGF scaffolds at 21 days, as well as significant improvements in survival in the short term at 2 and 7 days after implantation. Overall, our approach to sustaining EGFR signaling reduced MSC death in vivo and may be useful for future cell therapies where MSCs typically die on implantation.Stem cells are limited as tissue replacements owing to rapid death induced in the hostile wound environment. It has been found that restricting epidermal growth factor (EGF) receptor signaling to the membrane provides a survival advantage. This report elucidates a method to tether EGF to bone induction material to improve the survival of mesenchymal stem cells/multipotent stromal cells in vivo.
View details for DOI 10.5966/sctm.2015-0326
View details for PubMedID 27400798
High-Resolution Microfluidic Single-Cell Transcriptional Profiling Reveals Clinically Relevant Subtypes among Human Stem Cell Populations Commonly Utilized in Cell-Based Therapies.
Frontiers in neurology
2016; 7: 41-?
Mesenchymal stem cells remain of great interest in regenerative medicine due to their ability to home to sites of injury, differentiate into a variety of relevant lineages, and modulate inflammation and angiogenesis through paracrine activity. Many studies have found that despite the promise of MSC therapy, cell survival upon implant is highly limited and greatly reduces the therapeutic utility of MSCs. The matrikine Tenascin C, a protein expressed often at the edges of a healing wound, contains unique EGF-like repeats that are able to bind EGFR at low affinity and induce downstream pro-survival signaling without inducing receptor internalization. In this study, we utilized Tenascin C in a collagen/GAG-based polymer (Tpolymer) that has been shown to be beneficial for skin wound healing, incorporating human MSCs into the polymer prior to application to mouse punch biopsy wound beds. We found that the TPolymer was able to promote MSC survival out to 21 days in vivo, leading to associated improvements in wound healing such as dermal maturation and collagen content. This was most marked in a model of hypertrophic scarring, in which the scar formation was limited. This approach also reduced the inflammatory response in the wound bed, limiting CD3e+ cell invasion by approximately 50% in the early wound healing process, while increasing the numbers of endothelial cells during the first week of wound healing as well. Ultimately, this matrikine-based approach to improving MSC survival may be of great use across a variety of cell therapies utilizing matrices as delivery vehicles for cells.
View details for DOI 10.3727/096368916X692249
View details for PubMedID 27452449
Exercise induces stromal cell-derived factor-1a-mediated release of endothelial progenitor cells with increased vasculogenic function.
Plastic and reconstructive surgery
2015; 135 (2): 340e-50e
Stem cell therapies can promote neural repair and regeneration, yet controversy regarding optimal cell source and mechanism of action has slowed clinical translation, potentially due to undefined cellular heterogeneity. Single-cell resolution is needed to identify clinically relevant subpopulations with the highest therapeutic relevance. We combine single-cell microfluidic analysis with advanced computational modeling to study for the first time two common sources for cell-based therapies, human NSCs and MSCs. This methodology has the potential to logically inform cell source decisions for any clinical application.
View details for DOI 10.3389/fneur.2016.00041
View details for PubMedID 27047447
Human mesenchymal stem cells/multipotent stromal cells consume accumulated autophagosomes early in differentiation
STEM CELL RESEARCH & THERAPY
Endothelial progenitor cells have been shown to traffic to and incorporate into ischemic tissues, where they participate in new blood vessel formation, a process termed vasculogenesis. Previous investigation has demonstrated that endothelial progenitor cells appear to mobilize from bone marrow to the peripheral circulation after exercise. In this study, the authors investigate potential etiologic factors driving this mobilization and investigate whether the mobilized endothelial progenitor cells are the same as those present at baseline.Healthy volunteers (n = 5) performed a monitored 30-minute run to maintain a heart rate greater than 140 beats/min. Venous blood samples were collected before, 10 minutes after, and 24 hours after exercise. Endothelial progenitor cells were isolated and evaluated.Plasma levels of stromal cell-derived factor-1α significantly increased nearly two-fold immediately after exercise, with a nearly four-fold increase in circulating endothelial progenitor cells 24 hours later. The endothelial progenitor cells isolated following exercise demonstrated increased colony formation, proliferation, differentiation, and secretion of angiogenic cytokines. Postexercise endothelial progenitor cells also exhibited a more robust response to hypoxic stimulation.Exercise appears to mobilize endothelial progenitor cells and augment their function by means of stromal cell-derived factor 1α-dependent signaling. The population of endothelial progenitor cells mobilized following exercise is primed for vasculogenesis with increased capacity for proliferation, differentiation, secretion of cytokines, and responsiveness to hypoxia. Given the evidence demonstrating positive regenerative effects of exercise, this may be one possible mechanism for its benefits.
View details for DOI 10.1097/PRS.0000000000000917
View details for PubMedID 25626819
Noncontact, low-frequency ultrasound therapy enhances neovascularization and wound healing in diabetic mice.
Plastic and reconstructive surgery
2014; 134 (3): 402e-11e
Bone marrow mesenchymal stem cells/multipotent stromal cells (MSCs) are recruited to sites of injury and subsequently support regeneration through differentiation or paracrine activity. During periods of stress such as wound site implant or differentiation, MSCs are subjected to a variety of stressors that might activate pathways to improve cell survival and generate energy. In this study, we monitored MSC autophagy in response to the process of differentiation.MSC autophagosome structures were observed by using transmission electron microscopy and a tandem green fluorescent protein-red fluorescent protein autophagic flux reporter to monitor the mammalian microtubule-associated protein-1 light chain 3 (LC3) turnover in real time. MSCs were differentiated by using standard osteogenic and adipogenic media, and autophagy was examined during short-term and long-term differentiation via immunoblots for LC3I and II. Autophagy was modulated during differentiation by using rapamycin and bafilomycin treatments to disrupt the autophagosome balance during the early stages of the differentiation process, and differentiation was monitored in the long term by using Von Kossa and Oil Red O staining as well as quantitative polymerase chain reaction analysis of typical differentiation markers.We found that undifferentiated MSCs showed an accumulation of a large number of undegraded autophagic vacuoles, with little autophagic turnover. Stimulation of autophagy with rapamycin led to rapid degradation of these autophagosomes and greatly increased rough endoplasmic reticulum size. Upon induction of osteogenic differentiation, MSC expression of LC3II, a common autophagosome marker, was lost within 12 hours, consistent with increased turnover. However, during adipogenic differentiation, drug treatment to alter the autophagosome balance during early differentiation led to changes in differentiation efficiency, with inhibited adipocyte formation following rapamycin treatment and accelerated fat accumulation following autophagosome blockade by bafilomycin.Our findings suggest that MSCs exist in a state of arrested autophagy with high autophagosome accumulation and are poised to rapidly undergo autophagic degradation. This phenotype is highly sensitive, and a balance of autophagy appears to be key in efficient MSC differentiation and function, as evidenced by our results implicating autophagic flux in early osteogenesis and adipogenesis.
View details for DOI 10.1186/scrt530
View details for Web of Science ID 000349069100001
View details for PubMedID 25523618
Mechanotransduction and fibrosis
JOURNAL OF BIOMECHANICS
2014; 47 (9): 1997-2005
Chronic wounds are a major source of morbidity for patients and represent a significant health burden. Implementing noninvasive techniques that accelerate healing of these wounds would provide great benefit. Ultrasound appears to be an effective modality for the treatment of chronic wounds in humans. MIST Therapy is a noncontact, low-frequency ultrasound treatment delivered through a saline mist. A variety of mechanisms have been proposed to explain the efficacy of ultrasound therapy, but the underlying molecular and cellular pathways impacted by this technique remain unclear. The in vivo effect of noncontact, low-frequency ultrasound was therefore examined in a humanized excisional wound model.The treatment group received noncontact, low-frequency ultrasound therapy three times per week, whereas the control group received a standard dressing change. Wounds were photographed at regular intervals to calculate healing kinetics. Wound tissue was harvested and processed for histology, quantitative polymerase chain reaction, and enzyme-linked immunosorbent assay.The MIST group demonstrated significantly accelerated wound healing, with 17.3 days to wound closure compared with 24 days in the controls (p < 0.05). This improvement became evident by day 9, with healing evidenced by significantly decreased mean wound area relative to original size (68 percent versus 80 percent; p < 0.01). Expression of markers of neovascularization (stromal cell-derived factor 1, vascular endothelial growth factor, and CD31) was also increased in the wound beds of noncontact, low-frequency ultrasound-treated mice compared with controls.Noncontact, low-frequency ultrasound treatment improves neovascularization and wound closure rates in excisional wounds for diabetic mice, likely because of the stimulated release of angiogenic factors.
View details for DOI 10.1097/PRS.0000000000000467
View details for PubMedID 25158717
Diabetes impairs the angiogenic potential of adipose-derived stem cells by selectively depleting cellular subpopulations.
Stem cell research & therapy
2014; 5 (3): 79-?
Scarring and tissue fibrosis represent a significant source of morbidity in the United States. Despite considerable research focused on elucidating the mechanisms underlying cutaneous scar formation, effective clinical therapies are still in the early stages of development. A thorough understanding of the various signaling pathways involved is essential to formulate strategies to combat fibrosis and scarring. While initial efforts focused primarily on the biochemical mechanisms involved in scar formation, more recent research has revealed a central role for mechanical forces in modulating these pathways. Mechanotransduction, which refers to the mechanisms by which mechanical forces are converted to biochemical stimuli, has been closely linked to inflammation and fibrosis and is believed to play a critical role in scarring. This review provides an overview of our current understanding of the mechanisms underlying scar formation, with an emphasis on the relationship between mechanotransduction pathways and their therapeutic implications.
View details for DOI 10.1016/j.jbiomech.2014.03.031
View details for Web of Science ID 000338621900009
The Matrikine Tenascin-C Protects Multipotential Stromal Cells/Mesenchymal Stem Cells from Death Cytokines Such as FasL.
Tissue engineering. Part A
Pathophysiologic changes associated with diabetes impair new blood vessel formation and wound healing. Mesenchymal stem cells derived from adipose tissue (ASCs) have been used clinically to promote healing, although it remains unclear whether diabetes impairs their functional and therapeutic capacity.In this study, we examined the impact of diabetes on the murine ASC niche, as well as on the potential of isolated cells to promote neovascularization in vitro and in vivo. A novel single cell analytical approach was used to interrogate ASC heterogeneity and subpopulation dynamics in this pathologic setting.Our results demonstrate that diabetes alters the ASC niche in situ, and that diabetic ASCs are compromised in their ability to establish a vascular network both in vitro and in vivo. Moreover, these diabetic cells were ineffective in promoting soft tissue neovascularization and wound healing. Single cell transcriptional analysis identified a subpopulation of cells which was diminished in both type 1 and type 2 models of diabetes. These cells were characterized by the high expression of genes known to be important for new blood vessel growth.Perturbations in specific cellular subpopulations, visible only on a single cell level, represent a previously unreported mechanism for the dysfunction of diabetic ASCs. These data suggest that the utility of autologous ASCs for cell-based therapies in diabetic patients may be limited, and that interventions to improve cell function before application are warranted.
View details for DOI 10.1186/scrt468
View details for PubMedID 24943716
Production of Reactive Oxygen Species by Multipotent Stromal Cells/Mesenchymal Stem Cells Upon Exposure to Fas Ligand
2012; 21 (10): 2171-2187
Multipotential stromal cells/mesenchymal stem cells (MSCs) are attractive candidates for regenerative therapy due to the ability of these cells to differentiate and positively influence neighboring cells. However, on implantation for wound reconstruction, these cells are lost as they are challenged by nonspecific inflammation signals generated in the wound environment and in response to any implanted foreign body. We have previously shown that sustained and surface-restricted epidermal growth factor receptor (EGFR) signaling by a tethered form of its prototypal ligand EGF enhances survival of MSC in the presence of death cytokines such as FasL, serum deprivation, and low oxygen in vitro. This was proposed to be due to the plasma membrane restriction of EGFR signaling. Interestingly, during wound repair, an extracellular matrix (ECM) component Tenascin-C (TNC) containing EGF-like repeats (EGFL) and fibronectin-like repeats (FNL) is upregulated. A few of the 14 EGFL on each of the 6 arms, especially the 14th, bind as low-affinity/high-avidity ligands to EGFR causing sustained surface-restricted EGFR signaling. We queried whether signaling by this physiologically relevant EGFR matrikine also protects MSCs from FasL-induced death. MSCs grown on TNC and Collagen I (as TNC by itself is antiadhesive) displayed a survival advantage in the presence of FasL. TNC neither sequestered nor neutralized FasL; rather, the effects of survival were via cell signaling. This survival was dependent on TNC activating EGFR and downstream pathways of Erk and Akt through EGFL; to a much lesser extent, the FNL of TNC also contributed to survival. Taken together, these results suggest that providing MSCs with a nonimmunogenic naturally occurring ECM moiety such as TNC enhances their survival in the presence of death factors, and this advantage occurs via signaling through EGFR primarily and integrins only to a minor extent. This matrix component is proposed to supplement MSC delivery on the scaffolds to provide a survival advantage against death upon in vivo implantation.
View details for DOI 10.1089/ten.TEA.2012.0568
View details for PubMedID 23541003
Growth factor regulation of proliferation and survival of multipotential stromal cells
STEM CELL RESEARCH & THERAPY
Multipotent stromal cells (MSCs) can be differentiated into osteoblasts and chondrocytes, making these cells candidates to regenerate cranio-facial injuries and lesions in long bones. A major problem with cell replacement therapy, however, is the loss of transplanted MSCs at the site of graft. Reactive oxygen species (ROS) and nonspecific inflammation generated at the ischemic site have been hypothesized to lead to MSCs loss; studies in vitro show MSCs dying both in the presence of ROS or cytokines like FasL. We questioned whether MSCs themselves may be the source of these death inducers, specifically whether MSCs produce ROS under cytokine challenge. On treating MSCs with FasL, we observed increased ROS production within 2 h, leading to apoptotic death after 6 h of exposure to the cytokine. N-acetyl cysteine, an antioxidant, is able to protect MSCs from FasL-induced ROS production and subsequent ROS-dependent apoptosis, though the MSCs eventually succumb to ROS-independent death signaling. Epidermal growth factor (EGF), a cell survival factor, is able to protect cells from FasL-induced ROS production initially; however, the protective effect wanes with continued FasL exposure. In parallel, FasL induces upregulation of the uncoupling protein UCP2, the main uncoupling protein in MSCs, which is not abrogated by EGF; however, the production of ROS is followed by a delayed apoptotic cell death despite moderation by UCP2. FasL-induced ROS activates the stress-induced MAPK pathways JNK and p38MAPK as well as ERK, along with the activation of Bad, a proapoptotic protein, and suppression of survivin, an antiapoptotic protein; the latter two key modulators of the mitochondrial death pathway. FasL by itself also activates its canonical extrinsic death pathway noted by a time-dependent degradation of c-FLIP and activation of caspase 8. These data suggest that MSCs participate in their own demise due to nonspecific inflammation, holding implications for replacement therapies.
View details for DOI 10.3727/096368912X639035
View details for Web of Science ID 000313227300007
View details for PubMedID 22526333
Multipotential stromal cells (MSCs) have been touted to provide an alternative to conservative procedures of therapy, be it heart transplants, bone reconstruction, kidney grafts, or skin, neuronal and cartilage repair. A wide gap exists, however, between the number of MSCs that can be obtained from the donor site and the number of MSCs needed for implantation to regenerate tissue. Standard methods of MSC expansion being followed in laboratories are not fully suitable due to time and age-related constraints for autologous therapies, and transplant issues leave questions for allogenic therapies. Beyond these issues of sufficient numbers, there also exists a problem of MSC survival at the graft. Experiments in small animals have shown that MSCs do not persist well in the graft environment. Either there is no incorporation into the host tissue, or, if there is incorporation, most of the cells are lost within a month. The use of growth and other trophic factors may be helpful in counteracting these twin issues of MSC expansion and death. Growth factors are known to influence cell proliferation, motility, survival and morphogenesis. In the case of MSCs, it would be beneficial that the growth factor does not induce differentiation at an early stage since the number of early-differentiating progenitors would be very low. The present review looks at the effect of and downstream signaling of various growth factors on proliferation and survival in MSCs.
View details for DOI 10.1186/scrt32
View details for Web of Science ID 000292882600001
View details for PubMedID 20977782