Behavior Of Human Stem Cells Research Articles

Three-dimensional migration of human amniotic fluid stem cells involves mesenchymal and amoeboid modes and is regulated by mTORC1.

Three‐dimensional (3D) cell migration is an integral part of many physiologic processes. Although being well studied in the context of adult tissue homeostasis and cancer development, remarkably little is known about the invasive behavior of human stem cells. Using two different kinds of invasion assays, this study aimed at investigating and characterizing the 3D migratory capacity of human amniotic fluid stem cells (hAFSCs), a well‐established fetal stem cell type. Eight hAFSC lines were found to harbor pronounced potential to penetrate basement membrane (BM)‐like matrices. Morphological examination and inhibitor approaches revealed that 3D migration of hAFSCs involves both the matrix metalloprotease‐dependent mesenchymal, elongated mode and the Rho‐associated protein kinase‐dependent amoeboid, round mode. Moreover, hAFSCs could be shown to harbor transendothelial migration capacity and to exhibit a motility‐associated marker expression pattern. Finally, the potential to cross extracellular matrix was found to be induced by mTORC1‐activating growth factors and reduced by blocking mTORC1 activity. Taken together, this report provides the first demonstration that human stem cells exhibit mTORC1‐dependent invasive capacity and can concurrently make use of mesenchymal and amoeboid 3D cell migration modes, which represents an important step toward the full biological characterization of fetal human stem cells with relevance to both developmental research and stem cell‐based therapy.

Laminin α2 controls mouse and human stem cell behaviour during midbrain dopaminergic neuron development.

Development of the central nervous system requires coordination of the proliferation and differentiation of neural stem cells. Here, we show that laminin alpha 2 (lm-α2) is a component of the midbrain dopaminergic neuron (mDA) progenitor niche in the ventral midbrain (VM) and identify a concentration-dependent role for laminin α2β1γ1 (lm211) in regulating mDA progenitor proliferation and survival via a distinct set of receptors. At high concentrations, lm211-rich environments maintain mDA progenitors in a proliferative state via integrins α6β1 and α7β1, whereas low concentrations of lm211 support mDA lineage survival via dystroglycan receptors. We confirmed our findings in vivo, demonstrating that the VM was smaller in the absence of lm-α2, with increased apoptosis; furthermore, the progenitor pool was depleted through premature differentiation, resulting in fewer mDA neurons. Examination of mDA neuron subtype composition showed a reduction in later-born mDA neurons of the ventral tegmental area, which control a range of cognitive behaviours. Our results identify a novel role for laminin in neural development and provide a possible mechanism for autism-like behaviours and the brainstem hypoplasia seen in some individuals with mutations of LAMA2.

Biomimetic Conditioning of Human Dentin Using Citric Acid

IntroductionIn carious teeth, transforming growth factor beta 1 (TGF-β1) is released from the dentin matrix and possibly activated in an acidic environment. Conversely, EDTA solutions with a neutral to slightly alkaline pH are used in clinics to promote cell homing in regenerative endodontic procedures. We hypothesized that citric acid (CA) might be more beneficial. MethodsTGF-β1 release from human dentin disks conditioned with either 10% CA (pH = 2) or 17% EDTA (pH = 8) and the behavior of human stem cells toward such pretreated dentin were studied. The protein concentration in conditioning solutions after 10 minutes of dentin exposure was determined using a pH-independent slot blot technique. ResultsThere was a 5-fold higher concentration of the target protein in CA (382 ± 30 ng/disk) compared with EDTA (66 ± 3 ng/disk, P < .005). Using confocal laser scanning microscopy on immunofluorescent-labeled disks, we identified a high density of TGF-β1 in peritubular dentin after CA treatment. A migration assay showed that CA conditioning attracted significantly more stem cells toward the dentin after 24 hours compared with EDTA (P < .05) or phosphate-buffered saline (P < .005). To investigate whether the cell response to these dentin surfaces could be affected by different pretreatments, we cultured stem cells on conditioned dentin disks and found that CA had a significantly (P < .05) better effect than EDTA on cell attachment and cell survival. ConclusionsCA conditioning could be useful and may have significant benefits over current treatments.

Mediating human stem cell behaviour via defined fibrous architectures by melt electrospinning writing

The architecture within which cells reside is key to mediating their specific functions within the body. In this study, we use melt electrospinning writing (MEW) to fabricate cell micro-environments with various fibrous architectures to study their effect on human stem cell behaviour. We designed, built and optimised a MEW apparatus and used it to fabricate four different platform designs of 10.4 ± 2 μm fibre diameter, with angles between fibres on adjacent layers of 90°, 45°, 10° and R (random). Mechanical characterisation was conducted via tensile testing, and human skeletal stem cells (hSSCs) were seeded to scaffolds to study the effect of architecture on cell morphology and mechanosensing (nuclear YAP). Cell morphology was significantly altered between groups, with cells on 90° scaffolds having a lower aspect ratio, greater spreading, greater cytoskeletal tension and nuclear YAP expression. Long term cell culture studies were then conducted to determine the differentiation potential of scaffolds in terms of alkaline phosphatase activity, collagen and mineral production. Across these studies, an increased cell spreading in 3-dimensions is seen with decreasing alignment of architecture correlated with enhanced osteogenesis. This study therefore highlights the critical role of fibrous architecture in regulating stem cell behaviour with implications for tissue engineering and disease progression. Statement of SignificanceThis is the first study which has investigated the effect of controlled fibrous architectures fabricated via melt electrospinning writing on stem cell behaviour and differentiation. After optimising the fabrication process and characterising scaffolds via SEM and mechanical testing, skeletal stem cells were seeded onto fibrous scaffolds with various micro-architectures. These architectures drove cell shape changes resulting in architecture dependent nuclear YAP localisation, suggesting altered mechanosensing at early time points. In agreement with these early markers, long term cell culture studies revealed for the first time that a 90° fibrous architecture is optimal for the osteogenic differentiation of skeletal stem cells.

Surface Characterization and Human Stem Cell Behaviors of Zirconia Implant Disks Biomimetic-Treated in Simulated Body Fluid.

This study investigated the effects of biomimetic deposition on a zirconia surface in simulated body fluid (SBF) and assessed the proliferation and differentiation of human bone marrow mesenchymal stem cells on the SBF-treated zirconia disks. Corrected SBF was prepared according to Kokubo's recipe. Eighty yttrium oxide-stabilized tetragonal zirconia polycrystalline disks were prepared and divided into two groups: (1) the test group with SBF-treated disks and (2) the control group with nontreated disks. Zirconia disks were soaked in SBF for 1, 4, 7, and 14 days at 36.5°C, and the hydroxyapatite (HA) precipitation was verified by analyzing the surface morphology. For more in-depth validation of HA formation, the surface roughness, composition, and crystallization of the 7-day treated disks were analyzed. Human bone marrow mesenchymal stem cells were used to further evaluate cell proliferation, alkaline phosphatase activity, and osteoblast gene expression on the 7-day treated zirconia disks. Disks showed different surface morphologies after soaking for different time periods. As the SBF soaking time increased, the amount of HA coverage increased gradually, uniformly covering the disks by day 7. There was no difference in surface roughness between the two groups (P > .05). Cell proliferation was higher on the SBF-treated disks (P < .05). At 9 days, alkaline phosphatase activity was higher on the SBF-treated disks (P < .05). Alkaline phosphatase staining was significant on the SBF-treated disks (P < .05). A gene study revealed that alkaline phosphatase and osteocalcin showed higher expression in SBF-treated disks (P < .05); however, collagen type I and runt-related transcription factor 2 did not show significant differences between the two groups (P > .05). This study demonstrated that biomimetic deposition has an effect on the formation of HA on zirconia disks. The cell attachment, proliferation, and differentiation of SBF-treated zirconia disks was superior to that of nontreated disks, which indicates that SBF-treated zirconia implants have long-term clinical value.

Modulation of human mesenchymal stem cell behavior on ordered tantalum nanotopographies fabricated using colloidal lithography and glancing angle deposition.

Ordered surface nanostructures have attracted much attention in biotechnology and biomedical engineering because of their potential to modulate cell-surface interactions in a controllable manner. However, the ability to fabricate large area ordered nanostructures is limited because of high costs and low speed of fabrication. Here, we have fabricated ordered nanostructures with large surface areas (1.5 × 1.5 cm(2)) using a combination of facile techniques including colloidal self-assembly, colloidal lithography and glancing angle deposition (GLAD). Polystyrene (722 nm) colloids were self-assembled into a hexagonally close-packed (hcp) crystal array at the water-air interface, transferred on a biocompatible tantalum (Ta) surface and used as a mask to generate an ordered Ta pattern. The Ta was deposited by sputter coating through the crystal mask creating approximately 60-nm-high feature sizes. The feature size was further increased by approximately 200-nm-height respectively using GLAD, resulting in the fabrication of four different surfaces (FLAT, Ta60, GLAD100, and GLAD200). Cell adhesion, proliferation, and osteogenic differentiation of primary human adipose-derived stem cells (hADSCs) were studied on these ordered nanostructures for up to 2 weeks. Our results suggested that cell spreading, focal adhesion formation, and filopodia extension of hADSCs were inhibited on the GLAD surfaces, while the growth rate was similar between each surface. Immunostaining for type I collagen (COL1) and osteocalcin (OC) showed that there was higher osteogenic components deposited on the GLAD surfaces compared to the Ta60 and FLAT surfaces after 1 week of osteogenic culture. After 2 weeks of osteogenic culture, alkaline phosphatase (ALP) activity and the amount of calcium was higher on the GLAD surfaces. In addition, osteoblast-like cells were confluent on Ta60 and FLAT surfaces, whereas the GLAD surfaces were not fully covered suggesting that the cell-cell interactions are stronger than cell-substrate interactions on GLAD surfaces. Visible extracellular matrix deposits decorated the porous surface can be found on the GLAD surfaces. Depth profiling of surface components using a new Ar cluster source and X-ray photoelectron spectroscopy (XPS) showed that deposited extracellular matrix on GLAD surfaces is rich in nitrogen. The fabricated ordered surface nanotopographies have potential to be applied in diverse fields, and demonstrate that the behavior of human stem cells can be directed on these ordered nanotopographies, providing new knowledge for applications in biomaterials and tissue engineering.

Inferring human stem cell behaviour from epigenetic drift

Although conventional experimental manipulations are impractical, it may be possible to infer human stem cell fates by 'reading' histories recorded within their genomes. Genomes are almost perfect copies of copies, and ancestries may be surreptitiously recorded by replication errors that inevitably accumulate. The greater the number of divisions, the greater the number of replication errors ('a molecular clock hypothesis'). Mutations rarely occur during a lifetime, but DNA methylation patterns are also copied after DNA replication and measurably drift with ageing at certain CpG sites in mitotic tissues, such as the colon. Such passenger methylation pattern variation may effectively function as 'epigenetic' somatic cell mitotic clocks. Replication errors can only accumulate in long-lived stem cell lineages, so methylation pattern drift largely records stem cell behaviour. How methylation patterns may encode stem cell ancestries is illustrated with two types of small reproductive units--colon crypt niches with continuous genealogies, and hair follicles with punctuated genealogies. Potentially, the genealogy of any human cell may be inferred by 'reading' its genome.

Formation of human hepatocyte-like cells with different cellular phenotypes by human umbilical cord blood-derived cells in the human–rat chimeras

We took advantage of the proliferative and permissive environment of the developing pre-immune fetus to develop a noninjury human–rat xenograft small animal model, in which the in utero transplantation of low-density mononuclear cells (MNCs) from human umbilical cord blood (hUCB) into fetal rats at 9–11 days of gestation led to the formation of human hepatocyte-like cells (hHLCs) with different cellular phenotypes, as revealed by positive immunostaining for human-specific alpha-fetoprotein (AFP), cytokeratin 19 (CK19), cytokeratin 8 (CK8), cytokeratin 18 (CK18), and albumin (Alb), and with some animals exhibiting levels as high as 10.7% of donor-derived human cells in the recipient liver. More interestingly, donor-derived human cells stained positively for CD34 and CD45 in the liver of 2-month-old rat. Human hepatic differentiation appeared to partially follow the process of hepatic ontogeny, as evidenced by the expression of AFP gene at an early stage and albumin gene at a later stage. Human hepatocytes generated in this model retained functional properties of normal hepatocytes. In this xenogeneic system, the engrafted donor-derived human cells persisted in the recipient liver for at least 6 months after birth. Taken together, these findings suggest that the donor-derived human cells with different cellular phenotypes are found in the recipient liver and hHLCs hold biological activity. This humanized small animal model, which offers an in vivo environment more closely resembling the situations in human, provides an invaluable approach for in vivo investigating human stem cell behaviors, and further in vivo examining fundamental mechanisms controlling human stem cell fates in the future.

Generation of human/rat xenograft animal model for the study of human donor stem cell behaviorsin vivo

To accurately and realistically elucidate human stem cell behaviors in vivo and the fundamental mechanisms controlling human stem cell fates in vivo, which is urgently required in regenerative medicine and treatments for some human diseases, a surrogate human-rat chimera model was developed. Human-rat chimeras were achieved by in utero transplanting low-density mononuclear cells from human umbilical cord blood into the fetal rats at 9-11 d of gestation, and subsequently, a variety of methods, including flow cytometry, PCR as well as immunohistochemical assay, were used to test the human donor contribution in the recipients. Of 29 live-born recipients, 19 had the presence of human CD45(+) cells in peripheral blood (PB) detected by flow cytometry, while PCR analysis on genomic DNA from 11 different adult tissues showed that 14 selected from flow cytometry-positive 19 animals possessed of donor-derived human cell engraftment in multiple tissues (i.e. liver, spleen, thymus, heart, kidney, blood, lung, muscle, gut and skin) examined at the time of tissue collection, as confirmed by detecting human beta2-microglobulin expression using immunohistochemistry. In this xenogeneic system, the engrafted donor-derived human cells persisted in multiple tissues for at least 6 mo after birth. Moreover, transplanted human donor cells underwent site-specific differentiation into CK18-positive human cells in chimeric liver and CD45-positive human cells in chimeric spleen and thymus of recipients. Taken together, these findings suggest that we successfully developed human-rat chimeras, in which xenogeneic human cells exist up to 6 mo later. This humanized small animal model, which offers an in vivo environment more closely resembling to the situations in human, provides an invaluable and effective approach for in vivo investigating human stem cell behaviors, and further in vivo examining fundamental mechanisms controlling human stem cell fates in the future. The potential for new advances in our better understanding the living biological systems in human provided by investigators in humanized animals will remain promising.

Gene marking and gene therapy directed at primary hematopoietic cells

The past year has been a very active one in the field of gene transfer to hematopoietic targets, specifically stem cells and T cells. A number of clinical trials were published that both demonstrated progress as well as identified problems that investigators will face in trying to make the technology therapeutically applicable. Important laboratory and animal experiments focused on predictive models for human stem cell behavior, methods for culturing and expanding primitive cells ex vivo, immune responses against transgenes, in vitro and in vivo selection of transduced cells, and alternatives to standard retroviral vectors.