Myh11+ microvascular mural cells and derived mesenchymal stem cells promote retinal fibrosis.

Retinal diseases are frequently characterized by the accumulation of excessive scar tissue found throughout the neural retina. However, the pathophysiology of retinal fibrosis remains poorly understood, and the cell types that contribute to the fibrotic response are incompletely defined. Here, we show that myofibroblast differentiation of mural cells contributes directly to retinal fibrosis. Using lineage tracing technology, we demonstrate that after chemical ocular injury, Myh11+ mural cells detach from the retinal microvasculature and differentiate into myofibroblasts to form an epiretinal membrane. Inhibition of TGFßR attenuates Myh11+ retinal mural cell myofibroblast differentiation, and diminishes the subsequent formation of scar tissue on the surface of the retina. We demonstrate retinal fibrosis within a murine model of oxygen-induced retinopathy resulting from the intravitreal injection of adipose Myh11-derived mesenchymal stem cells, with ensuing myofibroblast differentiation. In this model, inhibiting TGFßR signaling does not significantly alter myofibroblast differentiation and collagen secretion within the retina. This work shows the complexity of retinal fibrosis, where scar formation is regulated both by TGFßR and non-TGFßR dependent processes involving mural cells and derived mesenchymal stem cells. It also offers a cautionary note on the potential deleterious, pro-fibrotic effects of exogenous MSCs once intravitreally injected into clinical patients.

In vivo imaging of hemodynamic redistribution and arteriogenesis across microvascular network.

OBJECTIVE: Arteriogenesis is an important mechanism that contributes to restoration of oxygen supply in chronically ischemic tissues, but remains incompletely understood due to technical limitations. This study presents a novel approach for comprehensive assessment of the remodeling pattern in a complex microvascular network containing multiple collateral microvessels. METHODS: We have developed a hardware-software integrated platform for quantitative, longitudinal, and label-free imaging of network-wide hemodynamic changes and arteriogenesis at the single-vessel level. By ligating feeding arteries in the mouse ear, we induced network-wide hemodynamic redistribution and localized arteriogenesis. The utility of this technology was demonstrated by studying the influence of obesity on microvascular arteriogenesis. RESULTS: Simultaneously monitoring the remodeling of competing collateral arterioles revealed a new, inverse relationship between initial vascular resistance and extent of arteriogenesis. Obese mice exhibited similar remodeling responses to lean mice through the first week, including diameter increase and flow upregulation in collateral arterioles. However, these gains were subsequently lost in obese mice. CONCLUSIONS: Capable of label-free, comprehensive, and dynamic quantification of structural and functional changes in the microvascular network in vivo, this platform opens up new opportunities to study the mechanisms of microvascular arteriogenesis, its implications in diseases, and approaches to pharmacologically rectify microvascular dysfunction.

Fenestration Improves Acellular Dermal Matrix Biointegration: An Investigation of Revascularization with Photoacoustic Microscopy.

BACKGROUND: Acellular dermal matrices have revolutionized alloplastic breast reconstruction. Furthering our knowledge of their biointegration will allow for improved design of these biomaterials. The ideal acellular dermal matrix for breast reconstruction would provide durable soft-tissue augmentation while undergoing rapid biointegration to promote physiologic elasticity and reduced infectious complications. The inclusion of fenestrations in their design is thought to promote the process of biointegration; however, the mechanisms underlying this theory have not been evaluated. METHODS: Biointegration of standard and fenestrated acellular dermal matrices was assessed with serial photoacoustic microscopic imaging, in a murine dorsal skinfold window chamber model specifically designed to recapitulate the microenvironment of acellular dermal matrix-assisted alloplastic breast reconstruction. Photoacoustic microscopy allows for a serial, real-time, noninvasive assessment of hemoglobin content and oxygen saturation in living tissues, generating high-resolution, three-dimensional maps of the nascent microvasculature within acellular dermal matrices. Confirmatory histologic and immunohistochemical assessments were performed at the terminal time point. RESULTS: Fenestrated acellular dermal matrices demonstrated increased fibroblast and macrophage lineage host cell infiltration, greater mean percentage surface area vascular penetration (21 percent versus 11 percent; p = 0.08), and greater mean oxygen saturation (13.5 percent versus 6.9 percent; p < 0.05) than nonfenestrated matrices by 2 weeks after implantation. By 21 days, host cells had progressed nearly 1 mm within the acellular dermal matrix fenestrations, resulting in significantly more vascularity across the top of the fenestrated matrix (3.8 vessels per high-power field versus 0.07 vessels per high-power field; p < 0.05). CONCLUSIONS: Inclusion of fenestrations in acellular dermal matrices improves the recellularization and revascularization that are crucial to biointegration of these materials. Future studies will investigate the optimal distance between fenestrations.

Modified VEGF-A mRNA induces sustained multifaceted microvascular response and accelerates diabetic wound healing.

Capable of mediating efficient transfection and protein production without eliciting innate immune responses, chemically modified mRNA holds great potential to produce paracrine factors at a physiologically beneficial level, in a spatiotemporally controlled manner, and with low toxicity. Although highly promising in cardiovascular medicine and wound healing, effects of this emerging therapeutic on the microvasculature and its bioactivity in disease settings remain poorly understood. Here, we longitudinally and comprehensively characterize microvascular responses to AZD8601, a modified mRNA encoding vascular endothelial growth factor A (VEGF-A), in vivo. Using multi-parametric photoacoustic microscopy, we show that intradermal injection of AZD8601 formulated in a biocompatible vehicle results in pronounced, sustained and dose-dependent vasodilation, blood flow upregulation, and neovessel formation, in striking contrast to those induced by recombinant human VEGF-A protein, a non-translatable variant of AZD8601, and citrate/saline vehicle. Moreover, we evaluate the bioactivity of AZD8601 in a mouse model of diabetic wound healing in vivo. Using a boron nanoparticle-based tissue oxygen sensor, we show that sequential dosing of AZD8601 improves vascularization and tissue oxygenation of the wound bed, leading to accelerated re-epithelialization during the early phase of diabetic wound healing.

Myeloid P2Y2 receptor promotes acute inflammation but is dispensable for chronic high-fat diet-induced metabolic dysfunction.

The purinergic receptor P2Y2 binds ATP to control chemotaxis of myeloid cells, and global P2Y2 receptor knockout mice are protected in models of acute inflammation. Chronic inflammation mediated by macrophages and other immune cells in adipose tissue contributes to the development of insulin resistance. Here, we investigate whether mice lacking P2Y2 receptors on myeloid cells are protected against acute and chronic inflammation. Wild-type mice were transplanted with either wild-type or P2Y2 receptor null bone marrow and treated with a sublethal dose of endotoxin as a model of acute inflammation, or fed a high-fat diet to induce obesity and insulin resistance as a model of chronic inflammation. P2Y2-/- chimeric mice were protected against acute inflammation. However, high-fat diet feeding induced comparable inflammation and insulin resistance in both WT and P2Y2-/- chimeric mice. Of note, confocal microscopy revealed significantly fewer crown-like structures, assemblies of macrophages around adipocytes, in P2Y2-/- chimeric mice compared to WT chimeric mice. We conclude that P2Y2 receptors on myeloid cells are important in mediating acute inflammation but are dispensable for the development of whole body insulin resistance in diet-induced obese mice.

Effects of Collagenase Digestion and Stromal Vascular Fraction Supplementation on Volume Retention of Fat Grafts.

OBJECTIVE: The use of autologous fat as a soft tissue filler has increased over the past decade in both reconstructive and aesthetic surgeries. Enhancement of autologous fat grafts with the addition of the stromal vascular fraction (SVF) has been reported to improve long-term volume retention. Stromal vascular fraction is most commonly isolated using enzymatic digestion, but it is unknown what effect the digestion process has on the adipocytes and SVF cells that comprise the graft. Some clinicians have reported use of enzymatically digested fat grafts to alter the physical properties of the tissue in specialized applications. We have previously reported that increasing collagenase digestion duration adversely affects the viability of adipocytes and SVF cells. Here, we aimed to determine if collagenase digestion of adipocytes before grafting is detrimental to long-term graft retention and if SVF supplementation can abrogate these potential deleterious effects. METHODS AND RESULTS: We used a published xenograft model in which human lipoaspirate was implanted into the scalp of immunocompromised mice to study the effects of collagenase digestion on in vivo graft survival after 12 weeks. We used 4 experimental groups: grafts composed of collagenase-digested and nondigested adipocytes (50-minute digestion) and grafts with and without SVF supplementation. We used microcomputed tomography to serially and noninvasively quantify graft volume, in conjunction with hematoxylin-eosin staining of histological cross-sections of implanted and excised grafts to assess overall tissue viability. We found that adipocytes that were collagenase-digested before implantation had significantly lower retention rates at 12 weeks and poorer tissue health, which was assessed by quantifying the number of intact adipocytes, the number of cystic formations, and by scoring the degree of inflammation and fibrosis. Further, we found that SVF supplementation of the digested grafts improved graft survival, but not to the level observed in undigested grafts. CONCLUSIONS: We conclude that collagenase digestion adversely affects the long-term volume retention of fat grafts, but that graft retention is improved by SVF supplementation. These experimental results can serve as an initial framework to further elucidate the reported efficacy and safety of using collagenase-digested fat grafts and SVF in the clinical setting.

Arteriogenesis in murine adipose tissue is contingent on CD68+ /CD206+ macrophages.

OBJECTIVE: The surgical transfer of skin, fat, and/or muscle from a donor site to a recipient site within the same patient is a widely performed procedure in reconstructive surgeries. A surgical pretreatment strategy that is intended to increase perfusion in the flap, termed "flap delay," is a commonly employed technique by plastic surgeons prior to flap transplantation. Here, we explored whether CD68+ /CD206+ macrophages are required for arteriogenesis within the flap by performing gain-of-function and loss-of-function studies in a previously published flap delay murine model. METHODS AND RESULTS: Local injection of M2-polarized macrophages into the flap resulted in an increase in collateral vessel diameter. Application of a thin biomaterial film loaded with a pharmacological agent (FTY720), which has been previously shown to recruit CD68+ /CD206+ macrophages to remodeling tissue, increased CD68+ /CD206+ cell recruitment and collateral vessel enlargement. Conversely, when local macrophage populations were depleted within the inguinal fat pad via clodronate liposome delivery, we observed fewer CD68+ cells accompanied by diminished collateral vessel enlargement. CONCLUSIONS: Our study underscores the importance of macrophages during microvascular adaptations that are induced by flap delay. These studies suggest a mechanism for a translatable therapeutic target that may be used to enhance the clinical flap delay procedure.

Oxygen Sensing Difluoroboron ß-Diketonate Polylactide Materials with Tunable Dynamic Ranges for Wound Imaging.

Difluoroboron ß-diketonate poly(lactic acid) materials exhibit both fluorescence (F) and oxygen sensitive room-temperature phosphorescence (RTP). Introduction of halide heavy atoms (Br and I) is an effective strategy to control the oxygen sensitivity in these materials. A series of naphthyl-phenyl (nbm) dye derivatives with hydrogen, bromide and iodide substituents were prepared for comparison. As nanoparticles, the hydrogen derivative was hypersensitive to oxygen (0-0.3%), while the bromide analogue was suited for hypoxia detection (0-3% O2). The iodo derivative, BF2nbm(I)PLA, showed excellent F to RTP peak separation and an 0-100% oxygen sensitivity range unprecedented for metal-free RTP emitting materials. Due to the dual emission and unconventionally long RTP lifetimes of these O2 sensing materials, a portable, cost-effective camera was used to quantify oxygen levels via lifetime and red/green/blue (RGB) ratiometry. The hypersensitive H dye was well matched to lifetime detection, simultaneous lifetime and ratiometric imaging was possible for the bromide analogue, whereas the iodide material, with intense RTP emission and a shorter lifetime, was suited for RGB ratiometry. To demonstrate the prospects of this camera/material design combination for bioimaging, iodide boron dye-PLA nanoparticles were applied to a murine wound model to detect oxygen levels. Surprisingly, wound oxygen imaging was achieved without covering (i.e. without isolating from ambient conditions, air). Additionally, would healing was monitored via wound size reduction and associated oxygen recovery, from hypoxic to normoxic. These single-component materials provide a simple tunable platform for biological oxygen sensing that can be deployed to spatially resolve oxygen in a variety of environments.

Paradoxical Adipose Hyperplasia and Cellular Effects After Cryolipolysis: A Case Report.

Cryolipolysis is a noninvasive technique for the reduction of subcutaneous adipose tissue by controlled, localized cooling, causing adipocyte apoptosis, reportedly without affecting surrounding tissue. Although cryolipolysis has a low incidence of adverse side effects 33 cases of paradoxical adipose hyperplasia (PAH) have been reported and the precise pathogenesis of PAH is poorly understood. This present case study of PAH aims to characterize the pathological changes in the adipose tissue of PAH on a cellular level by using multiple different assays [hematoxy lin and eosin staining, LIVE/DEAD staining, BODIPY(®) 558/568 C12 (4,4-Difluoro-5-(2-Thienyl)-4-Bora-3a,4a-Diaza-s-Indacene-3-dodecanoic acid) staining]. to identify the underlying mechanism of PAH and reduce the prevalence of PAH in the future. Tissue with PAH had fewer viable cells, significantly decreased quantities of interstitial cells (p = 0.04), and fewer vessels per adipose tissue area when compared to the control tissue. Adipocytes from the PAH tissue were on average slightly smaller than the control adipocytes. Adipocytes of PAH tissue had irregularly contoured edges when compared to the smooth, round edges of the control tissue. These findings from a neutral third party are contrary to prior reports from the inventors of this technique regarding effects of cryolipolysis on both the microvasculature and interstitial cells in adipose tissue. Our use of different assays to compare cryolipolysis-treated PAH tissue with untreated adipose tissue in the same patient showed adipose tissue that developed PAH was hypocellular and hypovascular. Contrary to prior reports from the inventors, cryolipolysis may cause vessel loss, which could lead to ischemia and/or hypoxia that further contributes to adipocyte death. LEVEL OF EVIDENCE 5: Risk.

Macrophage Recruitment and Polarization During Collateral Vessel Remodeling in Murine Adipose Tissue.

OBJECTIVE: During autologous flap transplantation for reconstructive surgeries, plastic surgeons use a surgical pre-treatment strategy called "flap delay," which entails ligating a feeding artery into an adipose tissue flap 10-14 days prior to transfer. It is believed that this blood flow alteration leads to vascular remodeling in the flap, resulting in better flap survival following transfer; however, the structural changes in the microvascular network are poorly understood. Here, we evaluate microvascular adaptations within adipose tissue in a murine model of flap delay. METHODS AND RESULTS: We used a murine flap delay model in which we ligated an artery supplying the inguinal fat pad. Although the extent of angiogenesis appeared minimal, significant diameter expansion of pre-existing collateral arterioles was observed. There was a 5-fold increase in recruitment of CX3CR1(+) monocytes to ligated tissue, a threefold increase in CD68(+) /CD206(+) macrophages in ligated tissue, a 40% increase in collateral vessel diameters supplying ligated tissue, and a 6-fold increase in the number of proliferating cells in ligated tissue. CONCLUSIONS: Our study describes microvascular adaptations in adipose in response to altered blood flow and underscores the importance of macrophages. Our data supports the development of therapies that target macrophages in order to enhance vascular remodeling in flaps.

Pannexin 1 is required for full activation of insulin-stimulated glucose uptake in adipocytes.

OBJECTIVE: Defective glucose uptake in adipocytes leads to impaired metabolic homeostasis and insulin resistance, hallmarks of type 2 diabetes. Extracellular ATP-derived nucleotides and nucleosides are important regulators of adipocyte function, but the pathway for controlled ATP release from adipocytes is unknown. Here, we investigated whether Pannexin 1 (Panx1) channels control ATP release from adipocytes and contribute to metabolic homeostasis. METHODS: We assessed Panx1 functionality in cultured 3T3-L1 adipocytes and in adipocytes isolated from murine white adipose tissue by measuring ATP release in response to known activators of Panx1 channels. Glucose uptake in cultured 3T3-L1 adipocytes was measured in the presence of Panx1 pharmacologic inhibitors and in adipocytes isolated from white adipose tissue from wildtype (WT) or adipocyte-specific Panx1 knockout (AdipPanx1 KO) mice generated in our laboratory. We performed in vivo glucose uptake studies in chow fed WT and AdipPanx1 KO mice and assessed insulin resistance in WT and AdipPanx1 KO mice fed a high fat diet for 12 weeks. Panx1 channel function was assessed in response to insulin by performing electrophysiologic recordings in a heterologous expression system. Finally, we measured Panx1 mRNA in human visceral adipose tissue samples by qRT-PCR and compared expression levels with glucose levels and HOMA-IR measurements in patients. RESULTS: Our data show that adipocytes express functional Pannexin 1 (Panx1) channels that can be activated to release ATP. Pharmacologic inhibition or selective genetic deletion of Panx1 from adipocytes decreased insulin-induced glucose uptake in vitro and in vivo and exacerbated diet-induced insulin resistance in mice. Further, we identify insulin as a novel activator of Panx1 channels. In obese humans Panx1 expression in adipose tissue is increased and correlates with the degree of insulin resistance. CONCLUSIONS: We show that Panx1 channel activity regulates insulin-stimulated glucose uptake in adipocytes and thus contributes to control of metabolic homeostasis.

Differential Effects of Processing Time and Duration of Collagenase Digestion on Human and Murine Fat Grafts.

BACKGROUND: Autologous fat graft retention is unpredictable, and mechanisms of optimization are poorly understood. Attempts at improving retention use collagenase experimentally and clinically to isolate the stromal vascular fraction to "enhance" fat grafts. However, no standardized duration for collagenase digestion or time following fat graft harvest has been established. This study investigates the effect of (1) time after fat graft harvest and (2) collagenase digestion time on interstitial cell and adipocyte viability in murine fat and human lipoaspirate. METHODS: Murine fat and human lipoaspirate were incubated ex vivo after harvest at room temperature for 120 minutes. Additional groups were incubated with collagenase for increasing 5-minute intervals from 30 to 60 minutes. Samples from each group were stained with BODIPY to quantify intact adipocytes and the LIVE/DEAD kit to quantify interstitial cell viability. RESULTS: With increased time after harvest, the number of intact adipocytes in murine fat and human lipoaspirate remained unchanged. Human interstitial cells were resistant to the effect of increased time ex vivo, whereas murine interstitial cells decreased in viability. In both populations, increased collagenase digestion time significantly decreased the number of viable adipocytes (murine, p ≤ 0.001; human, p ≤ 0.001) and interstitial cells (murine, p ≤ 0.001; human, p ≤ 0.001). CONCLUSIONS: Human and murine adipocytes and human interstitial cells appear resistant to deleterious effects of increasing time following harvest. However, murine interstitial cells are sensitive to increased time and prolonged collagenase digestion. These studies highlight the complex cellular components of fat grafts and how they respond differentially to time and collagenase digestion.

Pericytes derived from adipose-derived stem cells protect against retinal vasculopathy.

BACKGROUND: Retinal vasculopathies, including diabetic retinopathy (DR), threaten the vision of over 100 million people. Retinal pericytes are critical for microvascular control, supporting retinal endothelial cells via direct contact and paracrine mechanisms. With pericyte death or loss, endothelial dysfunction ensues, resulting in hypoxic insult, pathologic angiogenesis, and ultimately blindness. Adipose-derived stem cells (ASCs) differentiate into pericytes, suggesting they may be useful as a protective and regenerative cellular therapy for retinal vascular disease. In this study, we examine the ability of ASCs to differentiate into pericytes that can stabilize retinal vessels in multiple pre-clinical models of retinal vasculopathy. METHODOLOGY/PRINCIPAL FINDINGS: We found that ASCs express pericyte-specific markers in vitro. When injected intravitreally into the murine eye subjected to oxygen-induced retinopathy (OIR), ASCs were capable of migrating to and integrating with the retinal vasculature. Integrated ASCs maintained marker expression and pericyte-like morphology in vivo for at least 2 months. ASCs injected after OIR vessel destabilization and ablation enhanced vessel regrowth (16% reduction in avascular area). ASCs injected intravitreally before OIR vessel destabilization prevented retinal capillary dropout (53% reduction). Treatment of ASCs with transforming growth factor beta (TGF-ß1) enhanced hASC pericyte function, in a manner similar to native retinal pericytes, with increased marker expression of smooth muscle actin, cellular contractility, endothelial stabilization, and microvascular protection in OIR. Finally, injected ASCs prevented capillary loss in the diabetic retinopathic Akimba mouse (79% reduction 2 months after injection). CONCLUSIONS/SIGNIFICANCE: ASC-derived pericytes can integrate with retinal vasculature, adopting both pericyte morphology and marker expression, and provide functional vascular protection in multiple murine models of retinal vasculopathy. The pericyte phenotype demonstrated by ASCs is enhanced with TGF-ß1 treatment, as seen with native retinal pericytes. ASCs may represent an innovative cellular therapy for protection against and repair of DR and other retinal vascular diseases.

Hypoxic culture and in vivo inflammatory environments affect the assumption of pericyte characteristics by human adipose and bone marrow progenitor cells.

Previous studies have shown that exposure to a hypoxic in vitro environment increases the secretion of pro-angiogenic growth factors by human adipose-derived stromal cells (hASCs) [Cao Y, et al., Biochem Biophys Res Commun 332: 370-379, 2005; Kokai LE, et al., Plast Reconstr Surg 116: 1453-1460, 2005; Park BS, et al., Biomed Res (Tokyo) 31: 27-34, 2010; Rasmussen JG, et al., Cytotherapy 13: 318-328, 2010; Rehman J, et al., Circulation 109: 1292-1298, 2004]. Previously, it has been demonstrated that hASCs can differentiate into pericytes and promote microvascular stability and maintenance during angiogenesis in vivo (Amos PJ, et al., Stem Cells 26: 2682-2690, 2008; Traktuev DO, et al., Circ Res 102: 77-85, 2008). In this study, we tested the hypotheses that angiogenic induction can be increased and pericyte differentiation decreased by pretreatment of hASCs with hypoxic culture and that hASCs are similar to human bone marrow-derived stromal cells (hBMSCs) in these regards. Our data confirms previous studies showing that hASCs: 1) secrete pro-angiogenic proteins, which are upregulated following culture in hypoxia, and 2) migrate up gradients of PDGF-BB in vitro, while showing for the first time that a rat mesenteric model of angiogenesis induced by 48/80 increases the propensity of both hASCs and hBMSCs to assume perivascular phenotypes following injection. Moreover, culture of both cell types in hypoxia before injection results in a biphasic vascular length density response in this model of inflammation-induced angiogenesis. The effects of hypoxia and inflammation on the phenotype of adult progenitor cells impacts both the therapeutic and the basic science applications of the cell types, as hypoxia and inflammation are common features of natural and pathological vascular compartments in vivo.

A new method for in vivo visualization of vessel remodeling using a near-infrared dye.

OBJECTIVES: Vascular obstructive events can be partially compensated for by remodeling processes that increase vessel diameter and collateral tortuosity. However, methods for visualizing remodeling events in vivo and with temporal comparisons from the same animal remain elusive. METHODS: Using a novel infrared conjugated polyethylene glycol dye, we investigated the possibility of intravital vascular imaging of the mouse ear before and after ligation of the primary feeder artery. For comparison, we used two different mouse models known to have impaired vascular remodeling after ligation (i.e., aged and PAI-1(-/-) mice). The results obtained with the infrared dye were confirmed using immunofluorescence labeling of the ear microvasculature with confocal microscopy. RESULTS: After ligation, increases in vessel diameter (between 10% and 60%) and tortuosity (approximately 15%) were observed in C57Bl/6 mice using both the infrared dye and the immunofluorescence technique. However, aged C57Bl/6 and PAI-1(-/-) mice did not show vascular remodeling following ligation. CONCLUSIONS: Vascular remodeling can be visualized and accurately quantified using a new infrared dye in vivo. This analysis technique could be generally employed for quantitative investigations of changes in vascular remodeling.

Microsphere size effects on embryoid body incorporation and embryonic stem cell differentiation.

Differentiation of pluripotent embryonic stem cells (ESCs) in vitro via multicellular spheroids called embryoid bodies (EBs) is commonly performed to model aspects of early mammalian development and initiate differentiation of cells for regenerative medicine technologies. However, the three-dimensional nature of EBs poses unique challenges for directed ESC differentiation, including limited diffusion into EBs of morphogenic molecules capable of specifying cell fate. Degradable polymer microspheres incorporated within EBs can present morphogenic molecules to ESCs in a spatiotemporally controlled manner to more efficiently direct differentiation. In this study, the effect of microsphere size on incorporation into EBs and ESC differentiation in response to microsphere- mediated morphogen delivery were assessed. PLGA microspheres with mean diameters of 1, 3, or 11 microm were fabricated and mixed with ESCs during EB formation. Smaller microspheres were incorporated more efficiently throughout EBs than larger microspheres, and regardless of size, retained for at least 10 days of differentiation. Retinoic acid release from incorporated microspheres induced EB cavitation in a size-dependent manner, with smaller microspheres triggering accelerated and more complete cavitation than larger particles. These results demonstrate that engineering the size of microsphere delivery vehicles incorporated within stem cell environments can be used to modulate the course of differentiation.

Homogeneous and organized differentiation within embryoid bodies induced by microsphere-mediated delivery of small molecules.

Cell specification and tissue formation during embryonic development are precisely controlled by the local concentration and temporal presentation of morphogenic factors. Similarly, pluripotent embryonic stem cells can be induced to differentiate in vitro into specific phenotypes in response to morphogen treatment. Embryonic stem cells (ESCs) are commonly differentiated as 3D spheroids referred to as embryoid bodies (EBs); however, differentiation of cells within EBs is typically heterogeneous and disordered. In this study, we demonstrate that in contrast to soluble morphogen treatment, delivery of morphogenic factors directly within EB microenvironments in a spatiotemporally controlled manner using polymer microspheres yields homogeneous, synchronous and organized ESC differentiation. Degradable PLGA microspheres releasing retinoic acid were incorporated directly within EBs and induced the formation of cystic spheroids uniquely resembling the phenotype and structure of early streak mouse embryos (E6.75), with an exterior of FOXA2+ visceral endoderm enveloping an epiblast-like layer of OCT4+ cells. These results demonstrate that controlled morphogen presentation to stem cells using degradable microspheres more efficiently directs cell differentiation and tissue formation than simple soluble delivery methods and presents a unique route to study the spatiotemporal effects of morphogenic factors on embryonic developmental processes in vitro.