Stem Cell Multilineage Differentiation Research Articles

A hierarchical bilayer architecture for complex tissue regeneration

Engineering a complete, physiologically functional, periodontal complex structure remains a great clinical challenge due to the highly hierarchical architecture of the periodontium and coordinated regulation of multiple growth factors required to induce stem cell multilineage differentiation. Using biomimetic self-assembly and microstamping techniques, we construct a hierarchical bilayer architecture consisting of intrafibrillarly mineralized collagen resembling bone and cementum, and unmineralized parallel-aligned fibrils mimicking periodontal ligament. The prepared biphasic scaffold possesses unique micro/nano structure, differential mechanical properties, and growth factor-rich microenvironment between the two phases, realizing a perfect simulation of natural periodontal hard/soft tissue interface. The interconnected porous hard compartment with a Young's modulus of 1409.00 ± 160.83 MPa could induce cross-arrangement and osteogenic differentiation of stem cells in vitro, whereas the micropatterned soft compartment with a Young's modulus of 42.62 ± 4.58 MPa containing abundant endogenous growth factors, could guide parallel arrangement and fibrogenic differentiation of stem cells in vitro. After implantation in critical-sized complete periodontal tissue defect, the biomimetic bilayer architecture potently reconstructs native periodontium with the insertion of periodontal ligament fibers into newly formed cementum and alveolar bone by recruiting host mesenchymal stem cells and activating the transforming growth factor beta 1/Smad3 signaling pathway. Taken together, integration of self-assembly and microstamping strategies could successfully fabricate a hierarchical bilayer architecture, which exhibits great potential for recruiting and regulating host stem cells to promote synergistic regeneration of hard/soft tissues.

Nanocellulose-Reinforced Hydroxyapatite Nanobelt Membrane as a Stem Cell Multi-Lineage Differentiation Platform for Biomimetic Construction of Bioactive 3D Osteoid Tissue In Vitro.

Severe bone defects, especially accompanied by vascular and peripheral nerve injuries, remain a massive challenge. Most studies related to bone tissue engineering have focused on osteogenic differentiation of mesenchymal stem cells (MSCs), and ignored the formation of blood vessels and nerves in the newly generated bone owing to the lack of proper materials and methodology for tuning stem cells differentiated into osteogenic, neuronal, and endothelial cells (ECs) in the same scaffold system. Herein, a nanocellulose-reinforced hybrid membrane with good mechanical properties and control over biodegradation by assembling ultralong hydroxyapatite nanobelts in a bacterial nanocellulose hydrogel is designed and synthesized. Osteogenic, neuronal cells are successfully differentiated on this hybrid membrane. Based on the multi-lineage differentiation property of the membrane, a bioactive 3D osteoid tissue (osteogenic, neural, and ECs) is mimetically constructed in vitro using layer-by-layer culture and integration. The bone regeneration ability of the as-prepared bioactive osteoid tissue is assessed in vivo via heterotopic osteogenesis experiments for eight weeks. The rapid new bone growth and formation of blood capillaries and nerve fibers prove that the hybrid membrane can be universally applied as a stem cell multi-lineage differentiation platform, which has significant applications in bone tissue engineering.

Runx2+ Niche Cells Maintain Incisor Mesenchymal Tissue Homeostasis through IGF Signaling

SUMMARYStem cell niches provide a microenvironment to support the self-renewal and multi-lineage differentiation of stem cells. Cell-cell interactions within the niche are essential for maintaining tissue homeostasis. However, the niche cells supporting mesenchymal stem cells (MSCs) are largely unknown. Using single-cell RNA sequencing, we show heterogeneity among Gli1+ MSCs and identify a subpopulation of Runx2+/Gli1+ cells in the adult mouse incisor. These Runx2+/Gli1+ cells are strategically located between MSCs and transit-amplifying cells (TACs). They are not stem cells but help to maintain the MSC niche via IGF signaling to regulate TAC proliferation, differentiation, and incisor growth rate. ATAC-seq and chromatin immunoprecipitation reveal that Runx2 directly binds to Igfbp3 in niche cells. This Runx2-mediated IGF signaling is crucial for regulating the MSC niche and maintaining tissue homeostasis to support continuous growth of the adult mouse incisor, providing a model for analysis of the molecular regulation of the MSC niche.

LncRNA Functions as a New Emerging Epigenetic Factor in Determining the Fate of Stem Cells.

Pluripotent stem cells have broad applications in regenerative medicine and offer ideal models for understanding the biological process of embryonic development and specific diseases. Studies suggest that the self-renewal and multi-lineage differentiation of stem cells are regulated by a complex network consisting of transcription factors, chromatin regulators, signaling factors, and non-coding RNAs. It is of great interest to identify RNA regulatory factors that determine the fate of stem cells. Long non-coding RNA (lncRNA), a class of non-coding RNAs with more than 200 bp in length, has been shown to act as essential epigenetic regulators of stem cell pluripotency and specific lineage commitment. In this review, we focus on recent research progress related to the function and epigenetic mechanisms of lncRNA in determining the fate of stem cells, particularly pluripotency maintenance and lineage-specific differentiation. We discuss the role of the Oct4 and Sox2 promoter-interacting lncRNA as identified by Chromatin RNA In Situ reverse Transcription sequencing (CRIST-seq). Further understanding of their potential actions will provide a basis for the development of regenerative medicine for clinical application. This work offers comprehensive details and better understanding of the role of lncRNA in determining the fate of stem cells and paves the way for clinical stem cell applications.

Isolation, characterization and multi-lineage differentiation of stem cells from human exfoliated deciduous teeth.

The aim of the present study was to isolate stem cells from human exfoliated deciduous teeth (SHEDs) and identify their phenotypes and multi-lineage differentiation potential. Three SHED cell strains were successfully isolated from three exfoliated deciduous teeth from different human subjects using the outgrowth method. Flow cytometric analysis indicated that SHEDs displayed high expression of the mesenchymal cell markers CD73 and CD90 but low expression of the hematopoietic stem cell marker CD34. PCR analysis illustrated that SHEDs expressed the mesenchymal stem cell markers CD44, CD73 and CD90, the osteoblast markers Alpl, Runx2, CBFA1 and collagen I, the cartilage cell markers Col10a1 and Acan, the adipose cell markers PPARγ2 and LPL, and the neuronal stem cell marker Nestin. In vitro induction experiments demonstrated the potential of the SHEDs for osteogenic, adipogenic and neurogenic differentiation. These SHED cells may be useful for further stem cell research and future therapeutic applications.

ROS functions as an upstream trigger for autophagy to drive hematopoietic stem cell differentiation

Background and objectives: We have recently described a mechanistic action of autophagy on hematopoiesis in which autophagy sustains hematopoietic stem cell multilineage differentiation by direct targeting of intracellular Notch. However, the upstream signal that triggers autophagy to degrade Notch during hematopoiesis remains elusive. Methods: Conditional autophagy-essential gene Atg7 knockout mouse model is used for identifying signals regulating autophagy in the promotion of hematopoiesis. Results: We find here that generation of reactive oxygen species (ROS) is progressively increased during hematopoietic stem cell differentiation, and inhibition of ROS production was found to attenuate the differentiation of hematopoietic stem cells. In hematopoietic stem and progenitor cells (HSPCs) of wild-type mice, inhibition of ROS production downregulated autophagy activity but upregulated intracellular Notch and its downstream effectors. In contrast, in the HSPCs of autophagy fully defective mice, ROS inhibition did not alter myeloid differentiation, and hematopoietic stem cell differentiation to multi-lineages no longer responded to ROS inhibition. Discussion: The ROS-regulating hematopoiesis is mitochondrial origin, and this action depends on intact autophagy machinery capable of degrading intracellular Notch. Conclusion: ROS functions as an upstream signal in the autophagic promotion of hematopoietic stem cell differentiation.

Autophagy Sustains Hematopoiesis Through Targeting Notch.

Autophagy is required for hematopoietic stem cell multilineage differentiation, but the underlying mechanism is unknown. Using a conditional mouse model and human leukemia cells, we uncovered a mechanistic link between autophagy and hematopoietic stem cell differentiation. Loss of autophagy in mouse hematopoietic stem cells diminished the bone marrow generation of functional blood cells, in particular lymphocytes, and resulted in a leukemic phenotype and elevated Notch signaling. Physiological autophagy activity in mice was inversely correlated with Notch signaling during adult hematopoietic stem cell differentiation, while pathologically low autophagy was associated with upregulated Notch signaling in dysfunctional hematopoietic stem cells of acute leukemia patients. Furthermore, we show that autophagy directly degraded intracellular Notch, the active form of Notch receptor cleaved from the full-length Notch molecule by γ-secretase. Finally, we show that hematopoietic multilineage differentiation potential was restored in autophagy defective hematopoietic stem and progenitor cells when their Notch signaling was abrogated either pharmacologically with γ-secretase inhibitor DAPT or genetically with RNA interference of Notch effector RBPJ. Hence, we propose that autophagy sustains hematopoiesis by direct targeting Notch.

Bioprintable, cell-laden silk fibroin–gelatin hydrogel supporting multilineage differentiation of stem cells for fabrication of three-dimensional tissue constructs

Bioprinting has exciting prospects for printing three-dimensional (3-D) tissue constructs by delivering living cells with appropriate matrix materials. However, progress in this field is currently extremely slow due to limited choices of bioink for cell encapsulation and cytocompatible gelation mechanisms. Here we report the development of clinically relevant sized tissue analogs by 3-D bioprinting, delivering human nasal inferior turbinate tissue-derived mesenchymal progenitor cells encapsulated in silk fibroin–gelatin (SF–G) bioink. Gelation in this bioink was induced via in situ cytocompatible gelation mechanisms, namely enzymatic crosslinking by mushroom tyrosinase and physical crosslinking via sonication. Mechanistically, tyrosinases oxidize the accessible tyrosine residues of silk and/or gelatin into reactive o-quinone moieties that can either condense with each other or undergo nonenzymatic reactions with available amines of both silk and gelatin. Sonication alters the hydrophobic interaction and accelerates self-assembly of silk fibroin macromolecules to form β-sheet crystals, which physically crosslink the hydrogel. However, sonication has no effect on the conformation of gelatin. The effect of optimized rheology, secondary conformations of silk–gelatin bioink, temporally controllable gelation strategies and printing parameters were assessed to achieve maximum cell viability and multilineage differentiation of the encapsulated human nasal inferior turbinate tissue-derived mesenchymal progenitor cells. This strategy offers a unique path forward in the direction of direct printing of spatially customized anatomical architecture in a patient-specific manner.

Concise Review: Hematopoietic Stem Cell Aging, Life Span, and Transplantation

Self-renewal and multilineage differentiation of stem cells are keys to the lifelong homeostatic maintenance of tissues and organs. Hematopoietic aging, characterized by immunosenescence, proinflammation, and anemia, is attributed to age-associated changes in the number and function of hematopoietic stem cells (HSCs) and their microenvironmental niche. Genetic variants and factors regulating stem cell aging are correlatively or causatively associated with overall organismal aging and longevity. Translational use of HSCs for transplantation and gene therapy demands effective methods for stem cell expansion. Targeting the molecular pathways involved in HSC self-renewal, proliferation, and homing has led to enhanced expansion and engraftment of stem cells upon transplantation. HSC transplantation is less effective in elderly people, even though this is the demographic with the greatest need for this form of treatment. Thus, understanding the biological changes in the aging of stem cells as well as local and systematic environments will improve the efficacy of aged stem cells for regenerative medicine and ultimately facilitate improved health and life spans.

Different roles of TGF-β in the multi-lineage differentiation of stem cells

Stem cells are a population of cells that has infinite or long-term self-renewal ability and can produce various kinds of descendent cells. Transforming growth factor β (TGF-β) family is a superfamily of growth factors, including TGF-β1, TGF-β2 and TGF-β3, bone morphogenetic proteins, activin/inhibin, and some other cytokines such as nodal, which plays very important roles in regulating a wide variety of biological processes, such as cell growth, differentiation, cell death. TGF-β, a pleiotropic cytokine, has been proved to be differentially involved in the regulation of multi-lineage differentiation of stem cells, through the Smad pathway, non-Smad pathways including mitogen-activated protein kinase pathways, phosphatidylinositol-3-kinase/AKT pathways and Rho-like GTPase signaling pathways, and their cross-talks. For instance, it is generally known that TGF-β promotes the differentiation of stem cells into smooth muscle cells, immature cardiomyocytes, chondrocytes, neurocytes, hepatic stellate cells, Th17 cells, and dendritic cells. However, TGF-β inhibits the differentiation of stem cells into myotubes, adipocytes, endothelial cells, and natural killer cells. Additionally, TGF-β can provide competence for early stages of osteoblastic differentiation, but at late stages TGF-β acts as an inhibitor. The three mammalian isoforms (TGF-β1, 2 and 3) have distinct but overlapping effects on hematopoiesis. Understanding the mechanisms underlying the regulatory effect of TGF-β in the stem cell multi-lineage differentiation is of importance in stem cell biology, and will facilitate both basic research and clinical applications of stem cells. In this article, we discuss the current status and progress in our understanding of different mechanisms by which TGF-β controls multi-lineage differentiation of stem cells.

SiRNA Nanoparticle Functionalization of Nanostructured Scaffolds Enables Controlled Multilineage Differentiation of Stem Cells

The creation of complex tissues and organs is the ultimate goal in tissue engineering. Engineered morphogenesis necessitates spatially controlled development of multiple cell types within a scaffold implant. We present a novel method to achieve this by adhering nanoparticles containing different small-interfering RNAs (siRNAs) into nanostructured scaffolds. This allows spatial retention of the RNAs within nanopores until their cellular delivery. The released siRNAs were capable of gene silencing BCL2L2 and TRIB2, in mesenchymal stem cells (MSCs), enhancing osteogenic and adipogenic differentiation, respectively. This approach for enhancing a single type of differentiation is immediately applicable to all areas of tissue engineering. Different nanoparticles localized to spatially distinct locations within a single implant allowed two different tissue types to develop in controllable areas of an implant. As a consequence of this, we predict that complex tissues and organs can be engineered by the in situ development of multiple cell types guided by spatially restricted nanoparticles.

Microenvironments Engineered by Inkjet Bioprinting Spatially Direct Adult Stem Cells Toward Muscle- and Bone-Like Subpopulations

In vivo, growth factors exist both as soluble and as solid-phase molecules, immobilized to cell surfaces and within the extracellular matrix. We used this rationale to develop more biologically relevant approaches to study stem cell behaviors. We engineered stem cell microenvironments using inkjet bioprinting technology to create spatially defined patterns of immobilized growth factors. Using this approach, we engineered cell fate toward the osteogenic lineage in register to printed patterns of bone morphogenetic protein (BMP) 2 contained within a population of primary muscle-derived stem cells (MDSCs) isolated from adult mice. This patterning approach was conducive to patterning the MDSCs into subpopulations of osteogenic or myogenic cells simultaneously on the same chip. When cells were cultured under myogenic conditions on BMP-2 patterns, cells on pattern differentiated toward the osteogenic lineage, whereas cells off pattern differentiated toward the myogenic lineage. Time-lapse microscopy was used to visualize the formation of multinucleated myotubes, and immunocytochemistry was used to demonstrate expression of myosin heavy chain (fast) in cells off BMP-2 pattern. This work provides proof-of-concept for engineering spatially controlled multilineage differentiation of stem cells using patterns of immobilized growth factors. This approach may be useful for understanding cell behaviors to immobilized biological patterns and could have potential applications for regenerative medicine.