Scaffold-free, Human Mesenchymal Stem Cell-Based Tissue Engineered Blood Vessels.

Tissue-engineered blood vessels (TEBV) can serve as vascular grafts and may also play an important role in the development of organs-on-a-chip. Most TEBV construction involves scaffolding with biomaterials such as collagen gel or electrospun fibrous mesh. Hypothesizing that a scaffold-free TEBV may be advantageous, we constructed a tubular structure (1 mm i.d.) from aligned human mesenchymal cell sheets (hMSC) as the wall and human endothelial progenitor cell (hEPC) coating as the lumen. The burst pressure of the scaffold-free TEBV was above 200 mmHg after three weeks of sequential culture in a rotating wall bioreactor and perfusion at 6.8 dynes/cm(2). The interwoven organization of the cell layers and extensive extracellular matrix (ECM) formation of the hMSC-based TEBV resembled that of native blood vessels. The TEBV exhibited flow-mediated vasodilation, vasoconstriction after exposure to 1 μM phenylephrine and released nitric oxide in a manner similar to that of porcine femoral vein. HL-60 cells attached to the TEBV lumen after TNF-α activation to suggest a functional endothelium. This study demonstrates the potential of a hEPC endothelialized hMSC-based TEBV for drug screening.

A long non-coding RNA targets microRNA miR-34a to regulate colon cancer stem cell asymmetric division.

The roles of long non-coding RNAs (lncRNAs) in regulating cancer and stem cells are being increasingly appreciated. Its diverse mechanisms provide the regulatory network with a bigger repertoire to increase complexity. Here we report a novel LncRNA, Lnc34a, that is enriched in colon cancer stem cells (CCSCs) and initiates asymmetric division by directly targeting the microRNA miR-34a to cause its spatial imbalance. Lnc34a recruits Dnmt3a via PHB2 and HDAC1 to methylate and deacetylate the miR-34a promoter simultaneously, hence epigenetically silencing miR-34a expression independent of its upstream regulator, p53. Lnc34a levels affect CCSC self-renewal and colorectal cancer (CRC) growth in xenograft models. Lnc34a is upregulated in late-stage CRCs, contributing to epigenetic miR-34a silencing and CRC proliferation. The fact that lncRNA targets microRNA highlights the regulatory complexity of non-coding RNAs (ncRNAs), which occupy the bulk of the genome.

Implantation of mouse embryonic stem cell-derived cardiac progenitor cells preserves function of infarcted murine hearts.

Stem cell transplantation holds great promise for the treatment of myocardial infarction injury. We recently described the embryonic stem cell-derived cardiac progenitor cells (CPCs) capable of differentiating into cardiomyocytes, vascular endothelium, and smooth muscle. In this study, we hypothesized that transplanted CPCs will preserve function of the infarcted heart by participating in both muscle replacement and neovascularization. Differentiated CPCs formed functional electromechanical junctions with cardiomyocytes in vitro and conducted action potentials over cm-scale distances. When transplanted into infarcted mouse hearts, CPCs engrafted long-term in the infarct zone and surrounding myocardium without causing teratomas or arrhythmias. The grafted cells differentiated into cross-striated cardiomyocytes forming gap junctions with the host cells, while also contributing to neovascularization. Serial echocardiography and pressure-volume catheterization demonstrated attenuated ventricular dilatation and preserved left ventricular fractional shortening, systolic and diastolic function. Our results demonstrate that CPCs can engraft, differentiate, and preserve the functional output of the infarcted heart.

Combined Gene Therapy and Functional Tissue Engineering for the Treatment of Osteoarthritis

The pathogenesis of osteoarthritis is mediated in part by inflammatory cytokines including interleukin-1 (IL-1), which promote degradation of articular cartilage and prevent human mesenchymal stem cell (hMSC) chondrogenesis. We combined gene therapy and functional tissue engineering to develop engineered cartilage with immunomodulatory properties that allow chondrogenesis in the presence of pathologic levels of IL-1 by inducing overexpression of IL-1 receptor antagonist (IL-1Ra) in hMSCs via scaffold-mediated lentiviral gene delivery. A doxycycline-inducible vector was used to transduce hMSCs in monolayer or within 3D woven PCL scaffolds to enable tunable IL-1Ra production. In the presence of IL-1, IL-1Ra-expressing engineered cartilage produced cartilage-specific extracellular matrix, while resisting IL-1-induced upregulation of matrix metalloproteinases and maintaining mechanical properties similar to native articular cartilage. The ability of functional engineered cartilage to deliver tunable anti-inflammatory cytokines to the joint may enhance the long-term success of therapies for cartilage injuries or osteoarthritis.

Following this, we modified this anti-inflammatory engineered cartilage to incorporate rabbit MSCs and evaluated this therapeutic strategy in a pilot study in vivo in rabbit osteochondral defects. Rabbits were fed a custom doxycycline diet to induce gene expression in engineered cartilage implanted in the joint. Serum and synovial fluid were collected and the levels of doxycycline and inflammatory mediators were measured. Rabbits were euthanized 3 weeks following surgery and tissues were harvested for analysis. We found that doxycycline levels in serum and synovial fluid were too low to induce strong overexpression of hIL-1Ra in the joint and hIL-1Ra was undetectable in synovial fluid via ELISA. Although hIL-1Ra expression in the first few days local to the site of injury may have had a beneficial effect, overall a higher doxycycline dose and more readily transduced cell population would improve application of this therapy.

In addition to the 3D woven PCL scaffold, cartilage-derived matrix scaffolds have recently emerged as a promising option for cartilage tissue engineering. Spatially-defined, biomaterial-mediated lentiviral gene delivery of tunable and inducible morphogenetic transgenes may enable guided differentiation of hMSCs into both cartilage and bone within CDM scaffolds, enhancing the ability of the CDM scaffold to provide chondrogenic cues to hMSCs. In addition to controlled production of anti-inflammatory proteins within the joint, in situ production of chondro- and osteo-inductive factors within tissue-engineered cartilage, bone, or osteochondral tissue may be highly advantageous as it could eliminate the need for extensive in vitro differentiation involving supplementation of culture media with exogenous growth factors. To this end, we have utilized controlled overexpression of transforming growth factor-beta 3 (TGF-β3), bone morphogenetic protein-2 (BMP-2) or a combination of both factors, to induce chondrogenesis, osteogenesis, or both, within CDM hemispheres. We found that TGF-β3 overexpression led to robust chondrogenesis in vitro and BMP-2 overexpression led to mineralization but not accumulation of type I collagen. We also showed the development of a single osteochondral construct by combining tissues overexpressing BMP-2 (hemisphere insert) and TGF-β3 (hollow hemisphere shell) and culturing them together in the same media. Chondrogenic ECM was localized in the TGF-β3-expressing portion and osteogenic ECM was localized in the BMP-2-expressing region. Tissue also formed in the interface between the two pieces, integrating them into a single construct.

Since CDM scaffolds can be enzymatically degraded just like native cartilage, we hypothesized that IL-1 may have an even larger influence on CDM than PCL tissue-engineered constructs. Additionally, anti-inflammatory engineered cartilage implanted in vivo will likely affect cartilage and the underlying bone. There is some evidence that osteogenesis may be enhanced by IL-1 treatment rather than inhibited. To investigate the effects of an inflammatory environment on osteogenesis and chondrogenesis within CDM hemispheres, we evaluated the ability of IL-1Ra-expressing or control constructs to undergo chondrogenesis and osteogenesis in the prescence of IL-1. We found that IL-1 prevented chondrogenesis in CDM hemispheres but did not did not produce discernable effects on osteogenesis in CDM hemispheres. IL-1Ra-expressing CDM hemispheres produced robust cartilage-like ECM and did not upregulate inflammatory mediators during chondrogenic culture in the presence of IL-1.

A Randomized Phase II Crossover Study of Imatinib or Rituximab for Cutaneous Sclerosis after Hematopoietic Cell Transplantation.

PURPOSE: Cutaneous sclerosis occurs in 20% of patients with chronic graft-versus-host disease (GVHD) and can compromise mobility and quality of life. EXPERIMENTAL DESIGN: We conducted a prospective, multicenter, randomized, two-arm phase II crossover trial of imatinib (200 mg daily) or rituximab (375 mg/m(2) i.v. weekly × 4 doses, repeatable after 3 months) for treatment of cutaneous sclerosis diagnosed within 18 months (NCT01309997). The primary endpoint was significant clinical response (SCR) at 6 months, defined as quantitative improvement in skin sclerosis or joint range of motion. Treatment success was defined as SCR at 6 months without crossover, recurrent malignancy or death. Secondary endpoints included changes of B-cell profiles in blood (BAFF levels and cellular subsets), patient-reported outcomes, and histopathology between responders and nonresponders with each therapy. RESULTS: SCR was observed in 9 of 35 [26%; 95% confidence interval (CI); 13%-43%] participants randomized to imatinib and 10 of 37 (27%; 95% CI, 14%-44%) randomized to rituximab. Six (17%; 95% CI, 7%-34%) patients in the imatinib arm and 5 (14%; 95% CI, 5%-29%) in the rituximab arm had treatment success. Higher percentages of activated B cells (CD27(+)) were seen at enrollment in rituximab-treated patients who had treatment success (P = 0.01), but not in imatinib-treated patients. CONCLUSIONS: These results support the need for more effective therapies for cutaneous sclerosis and suggest that activated B cells define a subgroup of patients with cutaneous sclerosis who are more likely to respond to rituximab.