Amanda N. Steele

Regulating Stem Cell Secretome Using Injectable Hydrogels with In Situ Network Formation

A family of shear-thinning hydrogels for injectable encapsulation and long-term delivery (SHIELD) has been designed and synthesized with controlled in situ stiffening properties to regulate the stem cell secretome. The authors demonstrate that SHIELD with an intermediate stiffness (200-400 Pa) could significantly promote the angiogenic potential of human adipose-derived stem cells.

Isolation and trans-differentiation of mesenchymal stromal cells into smooth muscle cells: Utility and applicability for cell-sheet engineering.

BACKGROUND Bone marrow (BM)-derived mesenchymal stromal cells (MSCs) have shown potential to differentiate into various cell types, including smooth muscle cells (SMCs). The extracellular matrix (ECM) represents an appealing and readily available source of SMCs for use in tissue engineering. In this study, we hypothesized that the ECM could be used to induce MSC differentiation to SMCs for engineered cell-sheet construction. METHODS Primary MSCs were isolated from the BM of Wistar rats, transferred and cultured on dishes coated with 3 different types of ECM: collagen type IV (Col IV), fibronectin (FN), and laminin (LM). Primary MSCs were also included as a control. The proportions of SMC (a smooth muscle actin [aSMA] and SM22a) and MSC markers were examined with flow cytometry and Western blotting, and cell proliferation rates were also quantified. RESULTS Both FN and LM groups were able to induce differentiation of MSCs toward smooth muscle-like cell types, as evidenced by an increase in the proportion of SMC markers (aSMA; Col IV 42.3 ± 6.9%, FN 65.1 ± 6.5%, LM 59.3 ± 7.0%, Control 39.9 ± 3.1%; P = 0.02, SM22; Col IV 56.0 ± 7.7%, FN 74.2 ± 6.7%, LM 60.4 ± 8.7%, Control 44.9 ± 3.6%) and a decrease in that of MSC markers (CD105: Col IV 64.0 ± 5.2%, FN 57.6 ± 4.0%, LM 60.3 ± 7.0%, Control 85.3 ± 4.2%; P = 0.03). The LM group showed a decrease in overall cell proliferation, whereas FN and Col IV groups remained similar to control MSCs (Col IV, 9.0 ± 2.3%; FN, 9.8 ± 2.5%; LM, 4.3 ± 1.3%; Control, 9.8 ± 2.8%). CONCLUSIONS Our findings indicate that ECM selection can guide differentiation of MSCs into the SMC lineage. Fibronectin preserved cellular proliferative capacity while yielding the highest proportion of differentiated SMCs, suggesting that FN-coated materials may be facilitate smooth muscle tissue engineering.

Layered smooth muscle cell–endothelial progenitor cell sheets derived from the bone marrow augment postinfarction ventricular function

Objective: The angiogenic potential of endothelial progenitor cells (EPCs) may be limited by the absence of their natural biologic foundation, namely smooth muscle pericytes. We hypothesized that joint delivery of EPCs and smooth muscle cells (SMCs) in a novel, totally bone marrow–derived cell sheet will mimic the native architecture of a mature blood vessel and act as an angiogenic construct to limit post infarction ventricular remodeling. Methods: Primary EPCs and mesenchymal stem cells were isolated from bone marrow of Wistar rats. Mesenchymal stem cells were transdifferentiated into SMCs by culture on fibronectin‐coated culture dishes. Confluent SMCs topped with confluent EPCs were detached from an Upcell dish to create a SMC‐EPC bi‐level cell sheet. A rodent model of ischemic cardiomyopathy was then created by ligating the left anterior descending artery. Rats were randomized into 3 groups: cell sheet transplantation (n = 9), no treatment (n = 12), or sham surgery control (n = 7). Results: Four weeks postinfarction, mature vessel density tended to increase in cell sheet‐treated animals compared with controls. Cell sheet therapy significantly attenuated the extent of cardiac fibrosis compared with that of the untreated group (untreated vs cell sheet, 198 degrees [interquartile range (IQR), 151‐246 degrees] vs 103 degrees [IQR, 92‐113 degrees], P = .04). Furthermore, EPC‐SMC cell sheet transplantation attenuated myocardial dysfunction, as evidenced by an increase in left ventricular ejection fraction (untreated vs cell sheet vs sham, 33.5% [IQR, 27.8%‐35.7%] vs 45.9% [IQR, 43.6%‐48.4%] vs 59.3% [IQR, 58.8%‐63.5%], P = .001) and decreases in left ventricular dimensions. Conclusions: The bone marrow‐derived, spatially arranged SMC‐EPC bi‐level cell sheet is a novel, multilineage cellular therapy obtained from a translationally practical source. Interactions between SMCs and EPCs augment mature neovascularization, limit adverse remodeling, and improve ventricular function after myocardial infarction.

Angiogenesis precedes cardiomyocyte migration in regenerating mammalian hearts

Objective: Although the mammalian hearts ability to fully regenerate is debated, its potential to extensively repair itself is gaining support. We hypothesized that heart regeneration relies on rapid angiogenesis to support myocardial regrowth and sought to characterize the timeline for angiogenesis and cell proliferation in regeneration. Methods: One‐day‐old CD‐1 mice (P1, N = 60) underwent apical resection or sham surgery. Hearts were explanted at serial time points from 0 to 30 days postresection and analyzed with immunohistochemistry to visualize vessel ingrowth and cardiomyocyte migration into the resected region. Proliferating cells were labeled with 5‐ethynyl‐2′‐deoxyuridine injections 12 hours before explant. 5‐Ethynyl‐2′‐deoxyuridine–positive cells were counted in both the apex and remote areas of the heart. Massons trichrome was used to assess fibrosis. Results: By 30 days postresection, hearts regenerated with minimal fibrosis. Compared with sham surgery, apical resection stimulated a significant increase in proliferation of preexisting cardiomyocytes between 3 and 11 days after injury. Capillary migration into the apical thrombus was detected as early as 2 days postresection, with development of mature arteries by 5 days postresection. New vessels became perfused by 5 days postresection as evidenced by lectin injection. Vessel density and diameter significantly increased within the resected area over 21 days, and vessel ingrowth always preceded cardiomyocyte migration, with coalignment of most migrating cardiomyocytes with ingrowing vessels. Conclusions: Endothelial cells migrate into the apical thrombus early after resection, develop into functional arteries, and precede cardiomyocyte ingrowth during mammalian heart regeneration. This endogenous neonatal response emphasizes the importance of expeditious angiogenesis required for neomyogenesis.

An innovative biologic system for photon-powered myocardium in the ischemic heart

Solar-powered heart? Harnessing light to create myocardial renewable energy. Coronary artery disease is one of the most common causes of death and disability, afflicting more than 15 million Americans. Although pharmacological advances and revascularization techniques have decreased mortality, many survivors will eventually succumb to heart failure secondary to the residual microvascular perfusion deficit that remains after revascularization. We present a novel system that rescues the myocardium from acute ischemia, using photosynthesis through intramyocardial delivery of the cyanobacterium Synechococcus elongatus. By using light rather than blood flow as a source of energy, photosynthetic therapy increases tissue oxygenation, maintains myocardial metabolism, and yields durable improvements in cardiac function during and after induction of ischemia. By circumventing blood flow entirely to provide tissue with oxygen and nutrients, this system has the potential to create a paradigm shift in the way ischemic heart disease is treated.

Stem Cell Therapy: Healing or Hype?: Why Stem Cell Delivery Doesn’t Work

During the last 2 decades, there has been a surge in the development of stem cell therapies to treat numerous debilitating diseases. Cardiovascular disease is a leading target of this research because of the minimal proliferative and regenerative capabilities of the heart. These studies have quickly progressed to clinical trials; however, the initial enthusiasm has faded because outcomes from these studies have led to disappointing and inconsistent results.1 This viewpoint offers an explanation as to why stem cells have yet to demonstrate a significant benefit in patients having cardiovascular disease and how these challenges should be addressed. It is currently unknown which cell lineage provides the greatest potential in regenerative effects. While most cardiac clinical trials have delivered mesenchymal stem cells or bone marrow–derived stem cells (BMSCs), others have used adipose-derived stem cells (ASCs) and cardiac stem cells (CSCs).1,2 Yet, regardless of the cell type used, stem cell trials for cardiovascular diseases have not yielded clinically meaningful outcomes, though most have only been statistically powered to demonstrate feasibility and safety. Mesenchymal stem cells are advantageous because they can be delivered to patients without the need for immunosuppression and secrete numerous antiapoptotic and angiogenic growth factors.3 In 2014, 30 patients were enrolled in a multicenter, double-blind trial and randomized to receive an intramyocardial injection of 25 million mesenchymal stem cells or cell medium concurrent with left ventricular assist device implantation. The authors concluded that administration of mesenchymal stem cells was feasible and safe, with a trend toward functional efficacy.3 BMSCs are commonly administered in cardiac trials and are attractive because of their proven safety and paracrine effects.4 A BMSC trial published in 2013 randomized 200 patients with acute myocardial infarction to an open-labeled, controlled trial with 2 BMSC groups. These cells were administered via …

SDF 1-alpha Attenuates Myocardial Injury Without Altering the Direct Contribution of Circulating Cells

Stromal cell-derived factor 1-alpha (SDF) is a potent bone marrow chemokine capable of recruiting circulating progenitor populations to injured tissue. SDF has known angiogenic capabilities, but bone marrow-derived cellular contributions to tissue regeneration remain controversial. Bone marrow from DsRed-transgenic donors was transplanted into recipients to lineage-trace circulating cells after myocardial infarction (MI). SDF was delivered post-MI, and hearts were evaluated for recruitment and plasticity of bone marrow-derived populations. SDF treatment improved ventricular function, border zone vessel density, and CD31+ cell frequency post-MI. Bone marrow-derived endothelial cells were observed; these cells arose through both cell fusion and transdifferentiation. Circulating cells also adopted cardiomyocyte fates, but such events were exceedingly rare and almost exclusively resulted from cell fusion. SDF did not significantly alter the proportion of circulating cells that adopted non-hematopoietic fates. Mechanistic insight into the governance of circulating cells is essential to realizing the full potential of cytokine therapies.

A novel protein-engineered hepatocyte growth factor analog released via a shear-thinning injectable hydrogel enhances post-infarction ventricular function

In the last decade, numerous growth factors and biomaterials have been explored for the treatment of myocardial infarction (MI). While pre‐clinical studies have demonstrated promising results, clinical trials have been disappointing and inconsistent, likely due to poor translatability. In the present study, we investigate a potential myocardial regenerative therapy consisting of a protein‐engineered dimeric fragment of hepatocyte growth factor (HGFdf) encapsulated in a shear‐thinning, self‐healing, bioengineered hydrogel (SHIELD). We hypothesized that SHIELD would facilitate targeted, sustained intramyocardial delivery of HGFdf thereby attenuating myocardial injury and post‐infarction remodeling. Adult male Wistar rats (n = 45) underwent sham surgery or induction of MI followed by injection of phosphate buffered saline (PBS), 10 μg HGFdf alone, SHIELD alone, or SHIELD encapsulating 10 μg HGFdf. Ventricular function, infarct size, and angiogenic response were assessed 4 weeks post‐infarction. Treatment with SHIELD + HGFdf significantly reduced infarct size and increased both ejection fraction and borderzone arteriole density compared to the controls. Thus, sustained delivery of HGFdf via SHIELD limits post‐infarction adverse ventricular remodeling by increasing angiogenesis and reducing fibrosis. Encapsulation of HGFdf in SHIELD improves clinical translatability by enabling minimally‐invasive delivery and subsequent retention and sustained administration of this novel, potent angiogenic protein analog. Biotechnol. Bioeng. 2017;114: 2379–2389.

Injectable Bioengineered Hydrogel Therapy in the Treatment of Ischemic Cardiomyopathy

Opinion statementOver the past two decades, the field of cardiovascular medicine has seen the rapid development of multiple different modalities for the treatment of ischemic myocardial disease. Most research efforts have focused on strategies aimed at coronary revascularization, with significant technological advances made in percutaneous coronary interventions as well as coronary artery bypass graft surgery. However, recent research efforts have shifted towards ways to address the downstream effects of myocardial infarction on both cellular and molecular levels. To this end, the broad application of injectable hydrogel therapy after myocardial infarction has stimulated tremendous interest. In this article, we will review what hydrogels are, how they can be bioengineered in unique ways to optimize therapeutic potential, and how they can be used as part of a treatment strategy after myocardial infarction.