Jangwook P. Jung

Myocardial Tissue Engineering with Cells Derived from Human-Induced Pluripotent Stem Cells and a Native-Like, High-Resolution, 3-Dimensionally Printed Scaffold

Rationale: Conventional 3-dimensional (3D) printing techniques cannot produce structures of the size at which individual cells interact. Objective: Here, we used multiphoton-excited 3D printing to generate a native-like extracellular matrix scaffold with submicron resolution and then seeded the scaffold with cardiomyocytes, smooth muscle cells, and endothelial cells that had been differentiated from human-induced pluripotent stem cells to generate a human-induced pluripotent stem cell–derived cardiac muscle patch (hCMP), which was subsequently evaluated in a murine model of myocardial infarction. Methods and Results: The scaffold was seeded with ≈50 000 human-induced pluripotent stem cell–derived cardiomyocytes, smooth muscle cells, and endothelial cells (in a 2:1:1 ratio) to generate the hCMP, which began generating calcium transients and beating synchronously within 1 day of seeding; the speeds of contraction and relaxation and the peak amplitudes of the calcium transients increased significantly over the next 7 days. When tested in mice with surgically induced myocardial infarction, measurements of cardiac function, infarct size, apoptosis, both vascular and arteriole density, and cell proliferation at week 4 after treatment were significantly better in animals treated with the hCMPs than in animals treated with cell-free scaffolds, and the rate of cell engraftment in hCMP-treated animals was 24.5% at week 1 and 11.2% at week 4. Conclusions: Thus, the novel multiphoton-excited 3D printing technique produces extracellular matrix–based scaffolds with exceptional resolution and fidelity, and hCMPs fabricated with these scaffolds may significantly improve recovery from ischemic myocardial injury.

Solid organ fabrication: comparison of decellularization to 3D bioprinting

Solid organ fabrication is an ultimate goal of Regenerative Medicine. Since the introduction of Tissue Engineering in 1993, functional biomaterials, stem cells, tunable microenvironments, and high-resolution imaging technologies have significantly advanced efforts to regenerate in vitro culture or tissue platforms. Relatively simple flat or tubular organs are already in (pre)clinical trials and a few commercial products are in market. The road to more complex, high demand, solid organs including heart, kidney and lung will require substantive technical advancement. Here, we consider two emerging technologies for solid organ fabrication. One is decellularization of cadaveric organs followed by repopulation with terminally differentiated or progenitor cells. The other is 3D bioprinting to deposit cell-laden bio-inks to attain complex tissue architecture. We reviewed the development and evolution of the two technologies and evaluated relative strengths needed to produce solid organs, with special emphasis on the heart and other tissues of the cardiovascular system.

ECM-incorporated hydrogels cross-linked via native chemical ligation to engineer stem cell microenvironments.

Limiting the precise study of the biochemical impact of whole molecule extracellular matrix (ECM) proteins on stem cell differentiation is the lack of 3D in vitro models that can accommodate many different types of ECM. Here we sought to generate such a system while maintaining consistent mechanical properties and supporting stem cell survival. To this end, we used native chemical ligation to cross-link poly(ethylene glycol) macromonomers under mild conditions while entrapping ECM proteins (termed ECM composites) and stem cells. Sufficiently low concentrations of ECM were used to maintain constant storage moduli and pore size. Viability of stem cells in composites was maintained over multiple weeks. ECM of composites encompassed stem cells and directed the formation of distinct structures dependent on ECM type. Thus, we introduce a powerful approach to study the biochemical impact of multiple ECM proteins (either alone or in combination) on stem cell behavior.

Imaging cardiac extracellular matrices: a blueprint for regeneration

Once damaged, cardiac tissue does not readily repair and is therefore a primary target of regenerative therapies. One regenerative approach is the development of scaffolds that functionally mimic the cardiac extracellular matrix (ECM) to deliver stem cells or cardiac precursor populations to the heart. Technological advances in micro/nanotechnology, stem cell biology, biomaterials and tissue decellularization have propelled this promising approach forward. Surprisingly, technological advances in optical imaging methods have not been fully utilized in the field of cardiac regeneration. Here, we describe and provide examples to demonstrate how advanced imaging techniques could revolutionize how ECM-mimicking cardiac tissues are informed and evaluated.

A nondenatured, noncrosslinked collagen matrix to deliver stem cells to the heart

AIMS Stem cell transplantation holds promise as a therapeutic approach for the repair of damaged myocardial tissue. One challenge of this approach is efficient delivery and long-term retention of the stem cells. Although several synthetic and natural biomaterials have been developed for this purpose, the ideal formulation has yet to be identified. MATERIALS & METHODS Here we investigate the utility of a nondenatured, noncrosslinked, commercially available natural biomaterial (TissueMend(®) [TEI Biosciences, Boston, MA, USA]) for delivery of human mesenchymal stem cells (MSCs) to the murine heart. RESULTS We found that MSCs attached, proliferated and migrated within and out of the TissueMend matrix in vitro. Human MSCs delivered to damaged murine myocardium via the matrix (2.3 × 10(4) ± 0.8 × 10(4) CD73(+) cells/matrix) were maintained in vivo for 3 weeks and underwent at least three population doublings during that period (21.9 × 10(4) ± 14.4 × 10(4) CD73(+) cells/matrix). In addition, collagen within the TissueMend matrix could be remodeled by MSCs in vivo, resulting in a significant decrease in the coefficient of alignment of fibers (0.12 ± 0.12) compared with the matrix alone (0.28 ± 0.07), and the MSCs were capable of migrating out of the matrix and into the host tissue. CONCLUSION Thus, TissueMend matrix offers a commercially available, biocompatible and malleable vehicle for the delivery and retention of stem cells to the heart.

Single-Cell RNA-Seq of Bone Marrow-Derived Mesenchymal Stem Cells Reveals Unique Profiles of Lineage Priming.

The plasticity and immunomodulatory capacity of mesenchymal stem cells (MSCs) have spurred clinical use in recent years. However, clinical outcomes vary and many ascribe inconsistency to the tissue source of MSCs. Yet unconsidered is the extent of heterogeneity of individual MSCs from a given tissue source with respect to differentiation potential and immune regulatory function. Here we use single-cell RNA-seq to assess the transcriptional diversity of murine mesenchymal stem cells derived from bone marrow. We found genes associated with MSC multipotency were expressed at a high level and with consistency between individual cells. However, genes associated with osteogenic, chondrogenic, adipogenic, neurogenic and vascular smooth muscle differentiation were expressed at widely varying levels between individual cells. Further, certain genes associated with immunomodulation were also inconsistent between individual cells. Differences could not be ascribed to cycles of proliferation, culture bias or other cellular process, which might alter transcript expression in a regular or cyclic pattern. These results support and extend the concept of lineage priming of MSCs and emphasize caution for in vivo or clinical use of MSCs, even when immunomodulation is the goal, since multiple mesodermal (and even perhaps ectodermal) outcomes are a possibility. Purification might enable shifting of the probability of a certain outcome, but is unlikely to remove multilineage potential altogether.

Heterogeneous Differentiation of Human Mesenchymal Stem Cells in 3D Extracellular Matrix Composites

Abstract Extracellular matrix (ECM) proteins are structural elements of tissue and also potent signaling molecules. Previously, our laboratory showed that ECM of 2D coatings can trigger differentiation of bone marrow-derived mesenchymal stem cells (MSCs) into mesodermal lineages in an ECM-specific manner over 14 days, in some cases comparable to chemical induction. To test whether a similar effect was possible in a 3D, tissue-like environment, we designed a synthetic-natural biomaterial composite. The composite can present whole-molecule ECM proteins to cells, even those that do not spontaneously form hydrogels ex vivo, in 3D. To this end, we entrapped collagen type I, laminin-111, or fibronectin in ECM composites with MSCs and directly compared markers of mesodermal differentiation including cardiomyogenic (ACTC1), osteogenic (SPP1), adipogenic (PPARG), and chondrogenic (SOX9) in 2D versus 3D. We found the 3D condition largely mimicked the 2D condition such that the addition of type I collagen was the most potent inducer of differentiation to all lineages tested. One notable difference between 2D and 3D was pronounced adipogenic differentiation in 3D especially in the presence of exogenous collagen type I. In particular, PPARG gene expression was significantly increased ∼16-fold relative to chemical induction, in 3D and not in 2D. Unexpectedly, 3D engagement of ECM proteins also altered immunomodulatory function of MSCs in that expression of IL-6 gene was elevated relative to basal levels in 2D. In fact, levels of IL-6 gene expression in 3D composites containing exogenously supplied collagen type I or fibronectin were statistically similar to levels attained in 2D with tumor necrosis factor-α (TNF-α) stimulation and these levels were sustained over a 2-week period. Thus, this novel biomaterial platform allowed us to compare the biochemical impact of whole-molecule ECM proteins in 2D versus 3D indicating enhanced adipogenic differentiation and IL-6 expression of MSC in the 3D context. Exploiting the biochemical impact of ECM proteins on MSC differentiation and immunomodulation could augment the therapeutic utility of MSCs.

An integrated statistical model for enhanced murine cardiomyocyte differentiation via optimized engagement of 3D extracellular matrices.

The extracellular matrix (ECM) impacts stem cell differentiation, but identifying formulations supportive of differentiation is challenging in 3D models. Prior efforts involving combinatorial ECM arrays seemed intuitively advantageous. We propose an alternative that suggests reducing sample size and technological burden can be beneficial and accessible when coupled to design of experiments approaches. We predict optimized ECM formulations could augment differentiation of cardiomyocytes derived in vitro. We employed native chemical ligation to polymerize 3D poly (ethylene glycol) hydrogels under mild conditions while entrapping various combinations of ECM and murine induced pluripotent stem cells. Systematic optimization for cardiomyocyte differentiation yielded a predicted solution of 61%, 24%, and 15% of collagen type I, laminin-111, and fibronectin, respectively. This solution was confirmed by increased numbers of cardiac troponin T, α-myosin heavy chain and α-sarcomeric actinin-expressing cells relative to suboptimum solutions. Cardiomyocytes of composites exhibited connexin43 expression, appropriate contractile kinetics and intracellular calcium handling. Further, adding a modulator of adhesion, thrombospondin-1, abrogated cardiomyocyte differentiation. Thus, the integrated biomaterial platform statistically identified an ECM formulation best supportive of cardiomyocyte differentiation. In future, this formulation could be coupled with biochemical stimulation to improve functional maturation of cardiomyocytes derived in vitro or transplanted in vivo.

Single-cell RNA-seq reveals activation of unique gene groups as a consequence of stem cell-parenchymal cell fusion

Fusion of donor mesenchymal stem cells with parenchymal cells of the recipient can occur in the brain, liver, intestine and heart following transplantation. The therapeutic benefit or detriment of resultant hybrids is unknown. Here we sought a global view of phenotypic diversification of mesenchymal stem cell-cardiomyocyte hybrids and associated time course. Using single-cell RNA-seq, we found hybrids consistently increase ribosome components and decrease genes associated with the cell cycle suggesting an increase in protein production and decrease in proliferation to accommodate the fused state. But in the case of most other gene groups, hybrids were individually distinct. In fact, though hybrids can express a transcriptome similar to individual fusion partners, approximately one-third acquired distinct expression profiles in a single day. Some hybrids underwent reprogramming, expressing pluripotency and cardiac precursor genes latent in parental cells and associated with developmental and morphogenic gene groups. Other hybrids expressed genes associated with ontologic cancer sets and two hybrids of separate experimental replicates clustered with breast cancer cells, expressing critical oncogenes and lacking tumor suppressor genes. Rapid transcriptional diversification of this type garners consideration in the context of cellular transplantation to damaged tissues, those with viral infection or other microenvironmental conditions that might promote fusion.

Simple Monolayer Differentiation of Murine Cardiomyocytes via Nutrient Deprivation-Mediated Activation of β-Catenin

Methods to generate murine cardiomyocytes from pluripotent stem cells (PSCs) in vitro are resource and time intensive. All current protocols require exogenously provided soluble factors and almost all utilize embryoid body formation to modulate pathways associated with mesoderm specification and cardiomyocyte differentiation. Here, we developed a simple protocol without EBs and without exogenous soluble factors that enabled cardiomyocyte differentiation of a murine induced PSC line based on controlled nutrient deprivation in 2D monolayer cultures. We showed that this protocol reproducibly imposed metabolic stress and consequently modulated active β-catenin levels to yield functional cardiomyocytes. The yield of cardiomyocytes and calcium handling kinetics were comparable to existing approaches. However, this approach did not produce consistent results between murine PSC lines suggesting signaling pathways linking nutrient deprivation to β-catenin activation are not universally conserved and may be a remnant of the parent population from which the induced PSCs were derived.