Rationale Conventional three-dimensional (3D) printing techniques cannot produce structures of the size at which individual cells interact. Objective Here, we used multiphoton-excited, 3-dimensional printing (MPE-3DP) to generate a native-like, extracellular matrix (ECM) scaffold with submicron resolution, and then seeded the scaffold with cardiomyocytes (CMs), smooth-muscle cells (SMCs), and endothelial cells (ECs) that had been differentiated from human induced-pluripotent stem cells (iPSCs) to generate a human, iPSC-derived cardiac muscle patch (hCMP), which was subsequently evaluated in a murine model of myocardial infarction (MI). Methods and Results The scaffold was seeded with ~50,000 human, iPSC-derived CMs, SMCs, and ECs (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 MI, 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 MPE-3DP technique produces ECM-based scaffolds with exceptional resolution and fidelity, and hCMPs fabricated with these scaffolds may significantly improve recovery from ischemic myocardial injury.

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.

Mesenchymal stem cells (MSCs) spontaneously fuse with somatic cells in vivo, albeit rarely, and the fusion products are capable of tissue-specific function (mature trait) or proliferation (immature trait), depending on the microenvironment. That stem cells can be programmed, or somatic cells reprogrammed, in this fashion suggests that stem cell fusion holds promise as a therapeutic approach for the repair of damaged tissues, especially tissues not readily capable of functional regeneration, such as the myocardium. In an attempt to increase the frequency of stem cell fusion and, in so doing, increase the potential for cardiac tissue repair, we expressed the fusogen of the vesicular stomatitis virus (VSV-G) in human MSCs. We found VSV-G expressing MSCs (vMSCs) fused with cardiomyocytes (CMs) and these fusion products adopted a CM-like phenotype and morphology in vitro. In vivo, vMSCs delivered to damaged mouse myocardium via a collagen patch were able to home to the myocardium and fuse to cells within the infarct and peri-infarct region of the myocardium. This study provides a basis for the investigation of the biological impact of fusion of stem cells with CMs in vivo and illustrates how viral fusion proteins might better enable such studies.

Cancer cell fusion was suggested as a mechanism of metastasis about a century ago. Since then, many additional modes of material transfer (i.e., tunneling nanotubes, and exosomes) to generate cell hybrids have been identified. However, studies documenting spontaneous tumor hybrid formation in vivo as a mechanism that enables metastasis are still lacking. Here, we tested whether spontaneous hybrid formation in vivo contributes to bona fide metastatic tumors. We first used single cell RNASeq to analyze the gene expression profile of spontaneously formed cancer cell-stromal hybrids, and results revealed that hybrids exhibit a clustering pattern that is distinct from either parental cell and suggestive of substantial diversity of individual hybrids. Despite the newly gained diversity, hybrids can retain expression of critical genes of each parental cell. To assess the biological impact of cancer cell hybrids in vivo , we transfected murine mammary tumor cells, isolated from FVB/N-Tg(MMTV-PyVT)634Mul/J mice (PyVT) with Cre recombinase prior to injection to the murine fat pad of FVB.129S6(B6)- Gt(ROSA)26Sor tm 1 (Luc)Kael /J mice such that luciferase expression is induced with hybrid formation; luciferase expression was tracked for up to four months. We observed that hybrid formation occurs spontaneously in vivo and that a significantly higher number of hybrids reside in metastases compared to the primary tumor, supporting the possibility that hybrids can emerge from the primary tumor and proliferate to help create a new tumor at a distant site. Additional studies are now warranted to delineate the mechanisms of cancer cell hybrid transit to metastases since drugs to inhibit hybrid formation might prevent metastatic spread.