Differences In Differentiation Potential Research Articles

Mouse mandibular-derived osteoclast progenitors have differences in intrinsic properties compared with femoral-derived progenitors.

Craniofacial osteoclasts are essential for site-specific processes such as alveolar bone resorption, tooth eruption, and orthodontic tooth movement. Much of the current understanding of osteoclast development and function comes from studies using long bone-derived cells. Minimal investigation has been done to explore skeletal site differences. The overall goal of this study was to determine if mandibular- and femoral-derived osteoclasts represent distinct populations. To test this hypothesis, bone marrow cells were initially analyzed from the mandible and femur of 2-month-old mice. It was shown that mandibular-derived osteoclasts have enhanced size (mm2) compared with femoral-derived osteoclasts. Since bone marrow macrophages are a heterogenous population, we additionally selected for monocytes and demonstrated that mandibular-derived monocytes also form osteoclasts with increased size compared with femoral-derived monocytes. Osteoclast precursor populations from both skeletal sites were analyzed by flow cytometry. A newly described Ly6CHigh+ population as well as the Ly6Cint population was increased in the mandibular-derived cells. The difference in differentiation potential between monocyte cultures suggests that the increase in the Ly6CHigh+ population may explain the enhanced differentiation potential in mandibular-derived cells. Monocyte genes such as Pu.1, C/ebp-a, and Prdm1 are increased in expression in mandibular-derived monocytes compared with femoral-derived monocytes. As expected with enhanced differentiation, osteoclast genes including Nfatc1, Dc-stamp, Ctsk, and Rank are upregulated in mandibular-derived osteoclast precursors. Future studies will determine how changes in the environment of the mandible lead to changes in percentages of osteoclast progenitors and their differentiation potential.

Stabilization of hESCs in two distinct substates along the continuum of pluripotency.

A detailed understanding of the developmental substates of human pluripotent stem cells (hPSCs) is needed to optimize their use in cell therapy and for modeling early development. Genetic instability and risk of tumorigenicity of primed hPSCs are well documented, but a systematic isogenic comparison between substates has not been performed. We derived four hESC lines in naive human stem cell medium (NHSM) and generated isogenic pairs of NHSM and primed cultures. Through phenotypic, transcriptomic, and methylation profiling, we identified changes that arose during the transition to a primed substate. Although early NHSM cultures displayed naive characteristics, including greater proliferation and clonogenic potential compared with primed cultures, they drifted toward a more primed-like substate over time, including accumulation of genetic abnormalities. Overall, we show that transcriptomic and epigenomic profiling can be used to place human pluripotent cultures along a developmental continuum and may inform their utility for clinical and research applications.

Validation of a color deconvolution method to quantify MSC tri-lineage differentiation across species.

Mesenchymal stem cells (MSCs) are a promising candidate for both human and veterinary regenerative medicine applications because of their abundance and ability to differentiate into several lineages. Mesenchymal stem cells are however a heterogeneous cell population and as such, it is imperative that they are unequivocally characterized to acquire reproducible results in clinical trials. Although the tri-lineage differentiation potential of MSCs is reported in most veterinary studies, a qualitative evaluation of representative histological images does not always unambiguously confirm tri-lineage differentiation. Moreover, potential differences in differentiation capacity are not identified. Therefore, quantification of tri-lineage differentiation would greatly enhance proper characterization of MSCs. In this study, a method to quantify the tri-lineage differentiation potential of MSCs is described using digital image analysis, based on the color deconvolution plug-in (ImageJ). Mesenchymal stem cells from three species, i.e., bovine, equine, and porcine, were differentiated toward adipocytes, chondrocytes, and osteocytes. Subsequently, differentiated MSCs were stained with Oil Red O, Alcian Blue, and Alizarin Red S, respectively. Next, a differentiation ratio (DR) was obtained by dividing the area % of the differentiation signal by the area % of the nuclear signal. Although MSCs isolated from all donors in all species were capable of tri-lineage differentiation, differences were demonstrated between donors using this quantitative DR. Our straightforward, simple but robust method represents an elegant approach to determine the degree of MSC tri-lineage differentiation across species. As such, differences in differentiation potential within the heterogeneous MSC population and between different MSC sources can easily be identified, which will support further optimization of regenerative therapies.

Mesenchymal Stromal Cells from the Epidermis and Dermis of Psoriasis Patients: Morphology, Immunophenotype, Differentiation Patterns, and Regulation of T Cell Proliferation

Psoriasis is a skin disease characterized by hyperproliferation of keratinocytes and chronic inflammation. Mesenchymal stem/stromal cells (MSCs) exhibit an immunoregulatory function that can be altered in the skin of these patients. However, to date, the presence and functional capacity of MSCs in the dermis and epidermis of patients with psoriasis have not been fully established. In the present study, we evaluated the presence of MSCs in the skin of patients by obtaining adherent cells from the dermis and epidermis of lesional and nonlesional areas and characterizing them in a comparative manner with corresponding cells obtained from the dermis (HD-MSCs) and epidermis (HE-MSCs) of healthy donors. We determined whether the adherent cells had immunophenotypic profiles and differentiation potentials that were characteristic of MSCs. In addition, we analyzed their immunosuppression function by evaluating their capacity to decrease T cell proliferation. Our results indicate the presence of MSCs in the dermis and epidermis of healthy donors and patients with psoriasis; adherent cells from all skin sources exhibited MSC characteristics, such as expression of CD73, CD90, and CD105 markers and a lack of hematopoietic and endothelial marker expression. However, the cell populations obtained showed differences in differentiation potential toward adipogenic, osteogenic, and chondrogenic lineages. In addition, we observed a low MSC obtention frequency in nonlesional epidermal samples (NLE-MSCs), which also showed alterations in morphology and proliferation rate. Interestingly, MSCs from both the nonlesional dermis (NLD-MSCs) and lesional dermis (LD-MSCs) showed higher HLA class I antigen (HLA-I) expression than HD-MSCs. Moreover, NLD-MSCs showed a low T cell proliferation suppression capacity. In summary, this study demonstrates the presence of MSCs in the epidermis and dermis of patients with psoriasis and suggests that such cells may favor the inflammatory process and thus psoriatic lesion development through high HLA-I expression and low immunosuppression capacity.

Rapid Screening of the Endodermal Differentiation Potential of Human Pluripotent Stem Cells.

Human pluripotent stem cells (hPSCs) hold tremendous promise for regenerative medicine, disease modeling, toxicology screening, and developmental biology. These applications are hindered due to inherent differences in differentiation potential observed among different hPSC lines. This is particularly true for the differentiation of hPSCs toward the endodermal lineage. Several groups have developed methods to screen hPSCs for their endodermal differentiation potential (EP). Particularly notable studies include (i) the use of WNT3A expression as a predictive biomarker, (ii) an embryoid body-based screen, and (iii) a transcriptomics-based approach. We recently developed a rapid screen to access the EP of hPSCs solely based on morphological analysis. The screen takes 4 days to perform and yields results that are easy to interpret. As the screen is based on our recently developed small molecule protocol for hepatocyte like cell (HLC) differentiation of hPSCs, this method is extremely cost-effective compared to the aforementioned approaches. © 2017 by John Wiley & Sons, Inc.

Culture Methods and Choice of Cells Makes a Difference in Outcome of Tissue Engineering- a Perspective

This perspective reviews the effects of different tissue engineering techniques, for induced differentiation of human embryonic stem cells into pulmonary lineage specific cells, and validating cell based regenerative therapy to ameliorate pulmonary degeneration. The etiology of degeneration differs from disease to disease. Likewise, strategies to regenerate lost tissues in its heterogeneity, should also cater to the regeneration process. To customize the same, researchers ought to device strategies to repair, replace or regenerate, in the spatio- temporal format, healthy tissues in their heterogeneous multi-functional existence, such that when transplanted in vitro, they may find their niche and integrate seamlessly into the tissue system and manifest full-fledged functional competence in order to reverse the erstwhile effects of degeneration. Cell based lung repair or regeneration is unquestionably the most promising agenda of regenerative medicine. In one of our studies, human embryonic stem cell (hESC) line H7 was successfully differentiated into three non-ciliated alveolar epithelial cells. In another study, BJNhem19 and BJNhem20 stem cell lines were used to induce similar guided endodermal differentiation into pulmonary lineage cells. Both studies were done using similar protocols, where the hESCs were first plated on gamma irradiated mouse embryonic fibroblast (MEF) feeder cells and undifferentiated hESCs were taken through embroid body formation. They were then subjected to induction into lung specific cells, by defined growth factors in Small Airways Growth Medium (SAGM) and Bronchiolar Endothelial Growth Media (BEGM). The H7 cells were found to express, both intracellularly and on their surfaces, characteristic marker proteins. However, the BJNhem19 and BJNhem20 cells showed poor differential potential, and could not be successfully differentiated into lung- specific cells. This difference in differentiation potential may be due to several intrinsic limitations, like genetic heterogeneity, anomalous morphogen receptivity or dormant/ unresponsive cytoplasmic determinants. These observations indicate that the choice of cells may play an important factor in tissue engineering. This review addresses the different culture methods and conditions needed for guided differentiation of stem cells. This perspective also discusses culture conditions of mesenchymal stem cells from different origins and dendritic stem cells from bone marrow.

Culture Methods and Choice of Cells Makes a Difference in Outcome of Tissue Engineering - A Perspective

This perspective reviews the effects of different tissue engineering techniques, for induced differentiation of human embryonic stem cells into pulmonary lineage specific cells, and validating cell based regenerative therapy to ameliorate pulmonary degeneration. The etiology of degeneration differs from disease to disease. Likewise, strategies to regenerate lost tissues in its heterogeneity, should also cater to the regeneration process. To customize the same, researchers ought to device strategies to repair, replace or regenerate, in the spatio- temporal format, healthy tissues in their heterogeneous multi-functional existence, such that when transplanted in vitro, they may find their niche and integrate seamlessly into the tissue system and manifest full-fledged functional competence in order to reverse the erstwhile effects of degeneration. Cell based lung repair or regeneration is unquestionably the most promising agenda of regenerative medicine. In one of our studies, human embryonic stem cell line H7 was successfully differentiated into three non-ciliated alveolar epithelial cells. In another study, BJNhem19 and BJNhem20 stem cell lines were used to induce similar guided endodermal differentiation into pulmonary lineage cells. Both studies were done using similar protocols, where the hESCs were first plated on gamma irriadiated MEF feeder cells and undifferentiated hESCs were taken through embroid body formation. They were then subjected to induction into lung specific cells, by defined growth factors in Small Airways Growth Medium and Bronchiolar Endothelial Growth Media. The H7 cells were found to express, both intracellularly and on their surfaces, characteristic marker proteins. However, the BJNhem19 and BJNhem20 cells showed poor differential potential, and could not be successfully differentiated into lung- specific cells. This difference in differentiation potential may be due to several intrinsic limitations, like genetic heterogeneity, anomalous morphogen receptivity or dormant/ unresponsive cytoplasmic determinants. These observations indicate that the choice of cells may play an important factor in tissue engineering. This review addresses the different culture methods and conditions needed for guided differentiation of stem cells. This perspective also discusses culture conditions of mesenchymal stem cells from different origins and dendritic stem cells from bone marrow

Single cell sorting identifies progenitor cell population from full thickness bovine articular cartilage

To date, no approved clinical intervention successfully prevents the progressive degradation of injured articular cartilage that leads to osteoarthritis (OA). Stem/progenitor cell populations within tissues of diarthrodial joint have shown their therapeutic potential in treating OA. However, this potential has not been fully realized due in part to the heterogeneity of these subpopulations. Characterization of clonal populations derived from a single cell may help identify more homogenous stem/progenitor populations within articular cartilage. Moreover, chondrogenic potential of clonal populations from different zones could be further examined to elucidate their differential roles in maintaining articular cartilage homeostasis. We combined Fluorescence-activated cell sorting (FACS) and clonogenicity screening to identify stem/progenitor cells cloned from single cells. High-efficiency colony-forming cells (HCCs) were isolated, and evaluated for stem/progenitor cell characteristics. HCCs were also isolated from different zones of articular cartilage. Their function was compared by lineage-specific gene expression, and differentiation potential. A difference in colony-forming efficiency was observed in terms of colony sizes. HCCs were highly clonogenic and multipotent, and overexpressed stem/progenitor cell markers. Also, proliferation and migration associated genes were over-expressed in HCCs. HCCs showed zonal differences with deep HCCs more chondrogenic and osteogenic than superficial HCCs. Our approach is a simple yet practical way to identify homogeneous stem/progenitor cell populations with clonal origin. The discovery of progenitor cells demonstrates the intrinsic self-repairing potential of articular cartilage. Differences in differentiation potential may represent the distinct roles of superficial and deep zone stem/progenitor cells in the maintenance of articular cartilage homeostasis.

Premature thymic involution impairs let7 up-regulation in the transition from fetal to adult hematopoietic stem cells in the thymus (HEM4P.236)

Abstract The postnatal thymus is a highly efficient microenvironment for promoting T cell specification and differentiation. However, hematopoietic stem cells (HSCs) possess age-dependent differences in differentiation potential into T or B cells, with fetal-type HSCs having a higher potential for producing B cells. The switch between fetal- and adult-type HSCs occurs in the perinatal period, associated with a transition from Lin28b to Let7 expression. We found that in Foxn1lacZ/lacZ mutant mice, which undergo premature thymic involution beginning at 1-2 weeks postnatally, the ETP and DN1a/b subsets were immediately and progressively reduced, while thymic B cells showed a transient increase peaking at around 3-4 weeks. Heterochronic chimeras showed that the capacity to produce B cells in mutant thymus was dependent on the age of HSCs, rather than the age of thymus. We further demonstrate that in thymic progenitor B cells, while Lin28b down regulation occurred normally, Let7g up regulation was impaired, repressing differentiation and promoting proliferation of progenitor B cells in Foxn1lacZ/lacZ thymus. Changes in the thymic microenvironment included progressive down regulation of CCL25, accelerated reduction of DL4 and a transient increase in IL7. Taken together, these data show that the switch from fetal-type to adult-type HSCs within thymus is controlled by thymic microenvironment at multiple levels, including lymphoid progenitor entry, fate specification, and differentiation.

Comprehensive Comparison Of Gene Expression, Genomic DNA Methylation, and In Vitro Hematopoietic Differentiation Among Many Human iPS and ES Cell Lines

ObjectiveHematopoietic differentiation from human induced pluripotent stem (iPS)/embryonic stem (ES) cell attracts much attention due to its huge potential for regenerative medicine. As indicated by some earlier papers, there is large variation in differentiation potential among pluripotent stem cell (PSC) lines, and this is one of major concerns in clinical application of PSCs. If it becomes possible to predict which PSC line has high differentiation potential without real differentiation experiment, it would greatly contribute to clinical application of PSCs. Although some papers reported about presence of epigenetic memories of parental somatic cells in iPS cells, the amount of the influence on differentiation potential remains to be known. Furthermore, especially in studies using human PSCs, genetic difference among individual donors of iPS/ES cells seems to be large, thus the study using many PSC lines from many donors is warranted. To address these issues, we planned to collect data of many iPS/ES cell lines on genome-wide gene expression and genomic DNA methylation, and differentiation potentials of individual lines, and identify the factors which affected difference in differentiation potential among PSC lines. The final goal of this study is to create data base about gene expression and DNA methylation profile and differentiation potentials of many PSC lines. We believe that this dataset will allow us to predict differentiation potentials of individual PSC lines, and accelerate clinical application of PSC lines in hematology field. MethodWe utilized 39 iPS/ES lines (iPS 35 lines, ES 4 lines) in this study. The iPS cell lines were derived from dermal fibroblast (n = 16), cord blood (n = 3), peripheral blood (n = 10), keratinocyte (n = 3), and dental pulp cell (n = 3), and were generated by retrovirus vector (n = 9), episomal vector (n = 25), and sendai virus vector (n = 1). The iPS cells were derived from 15 donors, and the ES cells were derived from 4 donors. We assessed hematopoietic differentiation potential by investigating hematopoietic differentiation efficiency for the first 15 days from start of differentiation, and colony forming potential of hematopoietic precursor cells (CD34+CD38-CD43+lineage marker- population) generated from PSC lines using semi-solid methylcellulose based-media. In addition, we collected genome-wide mRNA expression and DNA methylation profile of PSC lines, parental lines of iPS cells, hematopoietic precursor cells generated from PSCs by using mRNA microarray, genomic methylation beads array, and next generation sequencers, and analyzed correlation of these data with differentiation potentials of individual PSC lines. ResultWe have found that there is large variation in hematopoietic differentiation efficiency and colony forming ability as reported previously. Genome-wide investigation of gene expression and genomic DNA methylation revealed that expression of some genes or some factors were significantly correlated with hematopoietic differentiation efficiency or colony forming ability of hematopoietic precursor cells. Importantly, the factors affecting differentiation efficiency for first 15 days and those affecting colony-forming ability were absolutely different. More importantly, by combining several factors discovered in this analysis, we can predict hematopoietic differentiation potential of individual iPS/ES cell lines regardless of what parental cell lines iPS cells are derived or whether it is an iPS cell or ES cell. ConclusionFrom genome-wide analysis of gene expression and genomic DNA methylation, and hematopoietic differentiation experiments, we discovered the factors that were associated with difference in differentiation potential among PSC lines. Now, we are focusing on investigating molecular mechanisms by which the discovered factors are responsible for the difference in hematopoietic differentiation potentials among PSC lines. We believe that our findings will contribute not only to clinical application of hematopoietic cells generated from human PSCs, but also to further understanding of human developmental hematopoiesis. Disclosures:No relevant conflicts of interest to declare.

Suppression of Cytochrome P450 Reductase Enhances Long-Term Hematopoietic Stem Cell Repopulation Efficiency in Mice

BackgroundBone marrow microenvironment (niche) plays essential roles in the fate of hematopoietic stem cells (HSCs). Intracellular and extracellular redox metabolic microenvironment is one of the critical factors for the maintenance of the niche. Cytochrome P450 reductase (CPR) is an obligate electron donor to all microsomal cytochrome P450 enzymes (P450 or CYP), and contributes to the redox metabolic process. However, its role in maintaining HSCs is unknown.ObjectiveTo examine the effects of low CPR expression on HSCs function using a mouse model of globally suppressed Cpr gene expression (Cpr Low, CL mice).MethodsHematopoietic cell subpopulations in bone marrow (BM) and peripheral blood (PB) from WT and CL mice were examined for their repopulation and differentiation ability upon BM competitive transplantation and enriched HSC (LKS+) transplantation. Effects of low CPR expression on hematopoiesis were examined by transplanting normal BM cells into CL recipients. Reactive oxygen species (ROS), cell cycle, and apoptosis in CL mice were analyzed by flow cytometry for DCF-DA fluorescence intensity, Ki67 protein, and Annexin-V, respectively.ResultsThe levels of ROS in BM cells, HPCs and HSCs were comparable between CL and WT mice. In comparison to WT mice, the number of LT-HSCs or ST-HSCs was lower in CL mice while CMPs, GMPs and MEPs in CL mice were higher than that in WT control. Competitive transplantation assay revealed enhanced repopulation capacity of HSCs with low CPR expression, but no difference in differentiation potential upon in vitro experiments. Furthermore, lymphoid differentiation of donor cells decreased while their myeloid differentiation increased under CL microenvironment although the overall level of donor hematopoietic repopulation was not significantly altered.ConclusionsOur studies demonstrate that suppressing CPR expression enhances the repopulation efficiency of HSCs and a low CPR expression microenvironment favors the differentiation of myeloid over lymphoid lineage cells.

Stem Cell and Tissue Engineering Applications in Orthopaedics and Musculoskeletal Medicine

Stem Cell and Tissue Engineering Applications in Orthopaedics and Musculoskeletal Medicine

Human Embryonic and Fetal Mesenchymal Stem Cells Differentiate toward Three Different Cardiac Lineages in Contrast to Their Adult Counterparts

Mesenchymal stem cells (MSCs) show unexplained differences in differentiation potential. In this study, differentiation of human (h) MSCs derived from embryonic, fetal and adult sources toward cardiomyocytes, endothelial and smooth muscle cells was investigated. Labeled hMSCs derived from embryonic stem cells (hESC-MSCs), fetal umbilical cord, bone marrow, amniotic membrane and adult bone marrow and adipose tissue were co-cultured with neonatal rat cardiomyocytes (nrCMCs) or cardiac fibroblasts (nrCFBs) for 10 days, and also cultured under angiogenic conditions. Cardiomyogenesis was assessed by human-specific immunocytological analysis, whole-cell current-clamp recordings, human-specific qRT-PCR and optical mapping. After co-culture with nrCMCs, significantly more hESC-MSCs than fetal hMSCs stained positive for α-actinin, whereas adult hMSCs stained negative. Furthermore, functional cardiomyogenic differentiation, based on action potential recordings, was shown to occur, but not in adult hMSCs. Of all sources, hESC-MSCs expressed most cardiac-specific genes. hESC-MSCs and fetal hMSCs contained significantly higher basal levels of connexin43 than adult hMSCs and co-culture with nrCMCs increased expression. After co-culture with nrCFBs, hESC-MSCs and fetal hMSCs did not express α-actinin and connexin43 expression was decreased. Conduction velocity (CV) in co-cultures of nrCMCs and hESC-MSCs was significantly higher than in co-cultures with fetal or adult hMSCs. In angiogenesis bioassays, only hESC-MSCs and fetal hMSCs were able to form capillary-like structures, which stained for smooth muscle and endothelial cell markers.Human embryonic and fetal MSCs differentiate toward three different cardiac lineages, in contrast to adult MSCs. Cardiomyogenesis is determined by stimuli from the cellular microenvironment, where connexin43 may play an important role.