Distinct Differentiation Potential Research Articles

Myelodysplastic Syndrome Hematopoietic Stem Cells Can Undergo Differentiation Trajectory Rewiring to Escape Venetoclax Vulnerability

Myelodysplastic syndromes (MDS) are incurable neoplasms that arise from early hematopoietic stem cells (HSCs). The standard of care for MDS is hypomethylating agent (HMA) therapy. However, the disease invariably fails these agents, resulting in fatal cytopenias or progression to acute myeloid leukemia (AML) with a dismal prognosis. We previously showed that the cellular hierarchy of MDS HSCs predicts the mechanisms of progression after HMA failure and can guide the design or choice of specific therapeutic approaches targeting these cells (Ganan-Gomez et al., Nature Medicine 2022). MDS HSCs in one of two differentiation states, long-term HSCs or lymphoid-primed multipotent progenitors, have a “common myeloid progenitor (CMP) pattern” or “granulocytic-monocytic progenitor (GMP) pattern” of differentiation. HSCs in these 2 differentiation states persist throughout the disease course and, driven by the recurrent activation of BCL2- or NF-κB-mediated survival pathways, respectively, expand at disease progression. In our preclinical studies, pharmacologically inhibiting these pathways depleted MDS HSCs and reduced tumor burden, which suggests that only patients with “CMP pattern” MDS may benefit from venetoclax-based therapies. Accordingly, patients with “CMP pattern” MDS, whose disease progressed after frontline HMA failure, enrolled in clinical trials of venetoclax-based therapy (NCT04160052, NCT04550442, or NCT04655755; n=28: 12 “CMP pattern” MDS and 16 “GMP pattern” MDS) had a shorter cumulative time to complete remission (P=0.012) and a longer relapse-free survival duration (14.0 months vs 5.2 months) than those with “GMP pattern” MDS did. Consistent with the hypothesis that venetoclax-based therapy selectively targets different HSC populations after HMA therapy failure, bone marrow (BM) HSC frequencies were significantly decreased at the time of remission after venetoclax-based therapy in “CMP pattern” MDS patients, but not in those with “GMP pattern” MDS. These results explain why “GMP pattern” MDS are resistant to venetoclax therapy. However, 8 of the 12 patients with “CMP pattern” MDS relapsed during venetoclax treatment and/or progressed to AML after an initial remission. In 2 patients with “CMP pattern” MDS, sequential flow cytometry and next generation sequencing analyses showed that the HSC hierarchy switched to “GMP pattern” MDS before venetoclax failure. This immunophenotypic hierarchical change was associated with the detection or expansion of STAG2- or RUNX1-mutant clones with isolated trisomy 8. To evaluate whether the immunophenotypic switch resulted from the distinct differentiation potential of early HSCs or from the aberrant expression of cell surface markers, we performed sequential single-cell RNA sequencing analyses of BM mononuclear cells isolated from the 2 “CMP pattern” MDS patients whose disease changed hierarchy before venetoclax failure. Our analyses revealed that after undergoing significant depletion during disease remission, HSCs expanded at therapy failure. Differential expression analysis showed that the expansion of STAG2- or RUNX1-mutant clones in the HSC compartment not only rewired MDS HSCs' differentiation state towards a granulo-monocytic-biased trajectory but also changed HSCs' survival dependency from BCL2-mediated anti-apoptotic pathways to TNFa-induced pro-survival NF-κB signaling, thus enabling HSCs to evade the cytotoxic effects of venetoclax (Fig. 1). The anti-apoptotic gene MCL1, a direct transcriptional target of NF-κB, was also significantly upregulated in HSCs at the time of progression, which suggests that the combination of venetoclax with an MCL1 inhibitor can overcome secondary failure to venetoclax. Together, these results reveal a novel mechanism of venetoclax resistance. HSCs under venetoclax therapy undergo survival pressure, which results in the expansion or acquisition of clones carrying specific mutations that change HSCs' survival dependencies (Fig. 2). Our study suggests that MDS patients receiving venetoclax-based therapy should be monitored at the molecular level and enrolled in clinical trials of other targeted agents before their disease undergoes clonal evolution and HSC hierarchical rewiring.

Osteogenic Differentiation Potential of Mesenchymal Stem Cells Using Single Cell Multiomic Analysis.

Mesenchymal stem cells (MSC) are multipotent stem cells that can differentiate into multiple cell types, including osteoblasts, chondrocytes, and adipocytes. Osteoblast differentiation is reduced during osteoporosis development, resulting in reduced bone formation. Further, MSC isolated from different donors possess distinct osteogenic capacity. In this study, we used single-cell multiomic analysis to profile the transcriptome and epigenome of MSC from four healthy donors. Data were obtained from ~1300 to 1600 cells for each donor. These cells were clustered into four groups, indicating that MSC from different donors have distinct chromatin accessible regulatory elements for regulating gene expression. To investigate the mechanism by which MSC undergo osteogenic differentiation, we used the chromatin accessibility data from the single-cell multiome data to identify individual-specific enhancer-promoter pairs and evaluated the expression levels and activities of the transcriptional regulators. The MSC from four donors showed distinct differentiation potential into osteoblasts. MSC of donor 1 showed the largest average motif activities, indicating that MSC from donor 1 was most likely to differentiate into osteoblasts. The results of our validation experiments were consistent with the bioinformatics prediction. We also tested the enrichment of genome-wide association study (GWAS) signals of several musculoskeletal disease traits in the patient-specific chromatin accessible regions identified in the single-cell multiome data, including osteoporosis, osteopenia, and osteoarthritis. We found that osteoarthritis-associated variants were only enriched in the regions identified from donor 4. In contrast, osteoporosis and osteopenia variants were enriched in regions from donor 1 and least enriched in donor 4. Since osteoporosis and osteopenia are related to the density of bone cells, the enrichment of variants from these traits should be correlated with the osteogenic potential of MSC. In summary, this study provides large-scale data to link regulatory elements with their target genes to study the regulatory relationships during the differentiation of mesenchymal stem cells and provide a deeper insight into the gene regulatory mechanism.

Regionally distinct progenitor cells in the lower airway give rise to neuroendocrine and multiciliated cells in the developing human lung

Using scRNA-seq and microscopy, we describe a cell that is enriched in the lower airways of the developing human lung and identified by the unique coexpression of SCGB3A2/SFTPB/CFTR. To functionally interrogate these cells, we apply a single-cell barcode-based lineage tracing method, called CellTagging, to track the fate of SCGB3A2/SFTPB/CFTR cells during airway organoid differentiation in vitro. Lineage tracing reveals that these cells have a distinct differentiation potential from basal cells, giving rise predominantly to pulmonary neuroendocrine cells and a subset of multiciliated cells distinguished by high C6 and low MUC16 expression. Lineage tracing results are supported by studies using organoids and isolated cells from the lower noncartilaginous airway. We conclude that SCGB3A2/SFTPB/CFTR cells are enriched in the lower airways of the developing human lung and contribute to the epithelial diversity and heterogeneity in this region.

Origin and diversification of fibroblasts from the sclerotome in zebrafish

Fibroblasts play an important role in maintaining tissue integrity by secreting components of the extracellular matrix and initiating response to injury. Although the function of fibroblasts has been extensively studied in adults, the embryonic origin and diversification of different fibroblast subtypes during development remain largely unexplored. Using zebrafish as a model, we show that the sclerotome, a sub-compartment of the somite, is the embryonic source of multiple fibroblast subtypes including tenocytes (tendon fibroblasts), blood vessel associated fibroblasts, fin mesenchymal cells, and interstitial fibroblasts. High-resolution imaging shows that different fibroblast subtypes occupy unique anatomical locations with distinct morphologies. Long-term Cre-mediated lineage tracing reveals that the sclerotome also contributes to cells closely associated with the axial skeleton. Ablation of sclerotome progenitors results in extensive skeletal defects. Using photoconversion-based cell lineage analysis, we find that sclerotome progenitors at different dorsal-ventral and anterior-posterior positions display distinct differentiation potentials. Single-cell clonal analysis combined with in vivo imaging suggests that the sclerotome mostly contains unipotent and bipotent progenitors prior to cell migration, and the fate of their daughter cells is biased by their migration paths and relative positions. Together, our work demonstrates that the sclerotome is the embryonic source of trunk fibroblasts as well as the axial skeleton, and local signals likely contribute to the diversification of distinct fibroblast subtypes.

Identification of distinct subpopulations of Gli1-lineage cells in the mouse mandible.

The Hedgehog pathway gene Gli1 has been proposed to mark a subpopulation of skeletal stem cells (SSCs) in craniofacial bone. Skeletal stem cells (SSCs) are multi-potent cells crucial for the development and homeostasis of bone. Recent studies on long bones have suggested that skeletal stem cells in endochondral or intramembranous ossification sites have different differentiation capacities. However, this has not been well-defined in neural crest derived bones. Generally, the long bones are derived from mesoderm and follow an endochondral ossification model, while most of the cranial bones are neural crest (NC) in origin and follow an intramembranous ossification model. The mandible is unique: It is derived from the neural crest lineage but makes use of both modes of ossification. Early in fetal development, the mandibular body is generated by intramembranous ossification with subsequent endochondral ossification forming the condyle. The identities and properties for SSCs in these two sites remain unknown. Here, we use genetic lineage tracing in mouse to identify cells expressing the Hedgehog responsive gene Gli1, which is thought to mark the tissue resident SSCs. We track the Gli1+ cells, comparing cells within the perichondrium to those in the periosteum covering the mandibular body. In juvenile mice, these have distinct differentiation and proliferative potential. We also assess the presence of Sox10+ cells, thought to mark neural crest stem cells, but find no substantial population associated with the mandibular skeleton, suggesting that Sox10+ cells have limited contribution to maintaining postnatal mandibular bone. All together, our study indicates that the Gli1+ cells display distinct and limited differentiation capacity dependent on their regional associations.

Unbalanced development and progressive repair in human early mosaic and chimeric embryos

With the advancement of genetics and genetic diagnosis, more and more human mosaics and chimeras and even unprecedented type of them have been identified. Especially in the era of universal prenatal screening, human mosaics and chimeras have often been revealed in the embryonic and fetal period with high accuracy. Although mosaicism and chimerism are abnormal in theory, some of them have no detectable effect on embryonic, fetal and postnatal growth and development and adulthood life, which increases difficulties being assessed during genetic counseling. In addition, the following intriguing observations on human mosaicism and chimerism are frequently reported. Continuous prenatal diagnoses indicate that the degree of fetal mosaicism is reduced in some subjects; Mosaicism and chimerism distribute differentially among tissues and organs of an individual; Cells with chromosomal abnormality gradually decrease or even disappear after birth, and are eventually replaced by normal cells. However, to our knowledge, we do not understand how these mosaicisaic and chimerisic phenomenon occur. Here, we propose a hypothesis of unbalanced development and progressive repair to elucidate the underlying mechanisms of these unique mosaic and chimeric phenomenon. The hypothesis is that unbalanced development and progressive repair of human mosaicism and chimerism are achieved by selective pressure and genetic control during development, clonal depletion associated with distinct apoptosis or differentiation potential and heterogoneic division and biased allocation. If this hypothesis is proven, these proposed mechanisms are of great clinical significance in the diagnosis and management of embryonic and fetal mosaicism or chimerism.

Skeletal stem cells: a game changer of skeletal biology and regenerative medicine?

Skeletal stem cells (SSCs) were originally discovered in the bone marrow stroma. They are capable of self-renewal and multilineage differentiation into osteoblasts, chondrocytes, adipocytes, and stromal cells. Importantly, these bone marrow SSCs localize in the perivascular region and highly express hematopoietic growth factors to create the hematopoietic stem cell (HSC) niche. Thus, bone marrow SSCs play pivotal roles in orchestrating osteogenesis and hematopoiesis. Besides the bone marrow, recent studies have uncovered diverse SSC populations in the growth plate, perichondrium, periosteum, and calvarial suture at different developmental stages, which exhibit distinct differentiation potential under homeostatic and stress conditions. Therefore, the current consensus is that a panel of region-specific SSCs collaborate to regulate skeletal development, maintenance, and regeneration. Here, we will summarize recent advances of SSCs in long bones and calvaria, with a special emphasis on the evolving concept and methodology in the field. We will also look into the future of this fascinating research area that may ultimately lead to effective treatment of skeletal disorders.

Identification of Human Breast Adipose Tissue Progenitors Displaying Distinct Differentiation Potentials and Interactions with Cancer Cells.

Breast adipose tissue (AT) participates in the physiological evolution and remodeling of the mammary gland due to its high plasticity. It is also a favorable microenvironment for breast cancer progression. However, information on the properties of human breast adipose progenitor cells (APCs) involved in breast physiology or pathology is scant. We performed differential enzymatic dissociation of human breast AT lobules. We isolated and characterized two populations of APCs. Here we report that these distinct breast APC populations selectively expressed markers suitable for characterization. The population preferentially expressing ALPL (MSCA1) showed higher adipogenic potential. The population expressing higher levels of INHBA and CD142 acquired myofibroblast characteristics upon TGF-β treatment and a myo-cancer-associated fibroblast profile in the presence of breast cancer cells. This population expressed the immune checkpoint CD274 (PD-L1) and facilitated the expansion of breast cancer mammospheres compared with the adipogenic population. Indeed, the breast, as with other fat depots, contains distinct types of APCs with differences in their ability to specialize. This indicates that they were differentially involved in breast remodeling. Their interactions with breast cancer cells revealed differences in the potential for tumor dissemination and estrogen receptor expression, and these differences might be relevant to improve therapies targeting the tumor microenvironment.

Schwann cells in the subcutaneous adipose tissue have neurogenic potential and can be used for regenerative therapies.

Stem cell therapies for nervous system disorders are hindered by a lack of accessible autologous sources of neural stem cells (NSCs). In this study, neural crest-derived Schwann cells are found to populate nerve fiber bundles (NFBs) residing in mouse and human subcutaneous adipose tissue (SAT). NFBs containing Schwann cells were harvested from mouse and human SAT and cultured in vitro. During in vitro culture, SAT-derived Schwann cells remodeled NFBs to form neurospheres and exhibited neurogenic differentiation potential. Transcriptional profiling determined that the acquisition of these NSC properties can be attributed to dedifferentiation processes in cultured Schwann cells. The emerging population of cells were termed SAT-NSCs because of their considerably distinct gene expression profile, cell markers, and differentiation potential compared to endogenous Schwann cells existing in vivo. SAT-NSCs successfully engrafted to the gastrointestinal tract of mice, migrated longitudinally and circumferentially within the muscularis, differentiated into neurons and glia, and exhibited neurochemical coding and calcium signaling properties consistent with an enteric neuronal phenotype. These cells rescued functional deficits associated with colonic aganglionosis and gastroparesis, indicating their therapeutic potential as a cell therapy for gastrointestinal dysmotility. SAT can be harvested easily and offers unprecedented accessibility for the derivation of autologous NSCs from adult tissues. Evidence from this study indicates that SAT-NSCs are not derived from mesenchymal stem cells and instead originate from Schwann cells within NFBs. Our data describe efficient isolation procedures for mouse and human SAT-NSCs and suggest that these cells have potential for therapeutic applications in gastrointestinal motility disorders.

CD45RA+CD62L- ILCs in human tissues represent a quiescent local reservoir for the generation of differentiated ILCs.

Innate lymphoid cells (ILCs) are highly plastic and predominantly mucosal tissue-resident cells that contribute to both homeostasis and inflammation depending on the microenvironment. The discovery of naïve-like ILCs suggests an ILC differentiation process that is akin to naïve T cell differentiation. Delineating the mechanisms that underlie ILC differentiation in tissues is crucial for understanding ILC biology in health and disease. Here, we showed that tonsillar ILCs expressing CD45RA lacked proliferative activity, indicative of cellular quiescence. CD62L distinguished two subsets of CD45RA+ ILCs. CD45RA+CD62L+ ILCs (CD62L+ ILCs) resembled circulating naïve ILCs because they lacked the transcriptional, metabolic, epigenetic, and cytokine production signatures of differentiated ILCs. CD45RA+CD62L- ILCs (CD62L- ILCs) were epigenetically similar to CD62L+ ILCs but showed a transcriptional, metabolic, and cytokine production signature that was more akin to differentiated ILCs. CD62L+ and CD62L- ILCs contained uni- and multipotent precursors of ILC1s/NK cells and ILC3s. Differentiation of CD62L+ and CD62L- ILCs led to metabolic reprogramming including up-regulation of genes associated with glycolysis, which was needed for their effector functions after differentiation. CD62L- ILCs with preferential differentiation capacity toward IL-22-producing ILC3s accumulated in the inflamed mucosa of patients with inflammatory bowel disease. These data suggested distinct differentiation potential of CD62L+ and CD62L- ILCs between tissue microenvironments and identified that manipulation of these cells is a possible approach to restore tissue-immune homeostasis.

The Occurrence of Malignancy in Trypanosoma brucei brucei by Rapid Passage in Mice.

Pleomorphic Trypanosoma brucei are best known for their tightly controlled cell growth and developmental program, which ensures their transmissibility and host fitness between the mammalian host and insect vector. However, after long-term adaptation in the laboratory or by natural evolution, monomorphic parasites can be derived. The origin of these monomorphic forms is currently unclear. Here, we produced a series of monomorphic trypanosome stocks by artificially syringe-passage in mice, creating snapshots of the transition from pleomorphism to monomorphism. We then compared these artificial monomorphic trypanosomes, alongside several naturally monomorphic T. evansi and T. equiperdum strains, with the pleomorphic T. brucei. In addition to failing to generate stumpy forms in animal bloodstream, we found that monomorphic trypanosomes from laboratory and nature exhibited distinct differentiation patterns, which are reflected by their distinct differentiation potential and transcriptional changes. Lab-adapted monomorphic trypanosomes could still be induced to differentiate, and showed only minor transcriptional differences to that of the pleomorphic slender forms but some accumulated differences were observed as the passages progress. All naturally monomorphic strains completely fail to differentiate, corresponding to their impaired differentiation regulation. We propose that the natural phenomenon of trypanosomal monomorphism is actually a malignant manifestation of protozoal cells. From a disease epidemiological and evolutionary perspective, our results provide evidence for a new way of thinking about the origin of these naturally monomorphic strains, the malignant evolution of trypanosomes may raise some concerns. Additionally, these monomorphic trypanosomes may reflect the quantitative and qualitative changes in the malignant evolution of T. brucei, suggesting that single-celled protozoa may also provide the most primitive model of cellular malignancy, which could be a primitive and inherent biological phenomenon of eukaryotic organisms from protozoans to mammals.

Intrahepatic cholangiocyte regeneration from an Fgf-dependent extrahepatic progenitor niche in a zebrafishmodel of Alagille Syndrome.

Alagille Syndrome (ALGS) is a congenital disorder caused by mutations in the Notch ligand gene JAGGED1, leading to neonatal loss of intrahepatic duct (IHD) cells and cholestasis. Cholestasis can resolve in certain patients with ALGS, suggesting regeneration of IHD cells. However, the mechanisms driving IHD cell regeneration following Jagged loss remains unclear. Here, we show that cholestasis due to developmental loss of IHD cells can be consistently phenocopied in zebrafish with compound jagged1b and jagged2b mutations or knockdown. Leveraging the transience of jagged knockdown in juvenile zebrafish, we find that resumption of Jagged expression leads to robust regeneration of IHD cells through a Notch-dependent mechanism. Combining multiple lineage tracing strategies with whole-liver three-dimensional imaging, we demonstrate that the extrahepatic duct (EHD) is the primary source of multipotent progenitors that contribute to the regeneration, but not to the development, of IHD cells. Hepatocyte-to-IHD cell transdifferentiation is possible but rarely detected. Progenitors in the EHD proliferate and migrate into the liver with Notch signaling loss and differentiate into IHD cells if Notch signaling increases. Tissue-specific mosaic analysis with an inducible dominant-negative Fgf receptor suggests that Fgf signaling from the surrounding mesenchymal cells maintains this extrahepatic niche by directly preventing premature differentiation and allocation of EHD progenitors to the liver. Indeed, transcriptional profiling and functional analysis of adult mouse EHD organoids uncover their distinct differentiation and proliferative potential relative to IHD organoids. Our data show that IHD cells regenerate upon resumption of Jagged/Notch signaling, from multipotent progenitors originating from an Fgf-dependent extrahepatic stem cell niche. We posit that if Jagged/Notch signaling is augmented, through normal stochastic variation, gene therapy, or a Notch agonist, regeneration of IHD cells in patients with ALGS may be enhanced.

Tailored generation of insulin producing cells from canine mesenchymal stem cells derived from bone marrow and adipose tissue

The trend of regenerative therapy for diabetes in human and veterinary practices has conceptually been proven according to the Edmonton protocol and animal models. Establishing an alternative insulin-producing cell (IPC) resource for further clinical application is a challenging task. This study investigated IPC generation from two practical canine mesenchymal stem cells (cMSCs), canine bone marrow-derived MSCs (cBM-MSCs) and canine adipose-derived MSCs (cAD-MSCs). The results illustrated that cBM-MSCs and cAD-MSCs contain distinct pancreatic differentiation potential and require the tailor-made induction protocols. The effective generation of cBM-MSC-derived IPCs needs the integration of genetic and microenvironment manipulation using a hanging-drop culture of PDX1-transfected cBM-MSCs under a three-step pancreatic induction protocol. However, this protocol is resource- and time-consuming. Another study on cAD-MSC-derived IPC generation found that IPC colonies could be obtained by a low attachment culture under the three-step induction protocol. Further, Notch signaling inhibition during pancreatic endoderm/progenitor induction yielded IPC colonies through the trend of glucose-responsive C-peptide secretion. Thus, this study showed that IPCs could be obtained from cBM-MSCs and cAD-MSCs through different induction techniques. Also, further signaling manipulation studies should be conducted to maximize the protocol’s efficiency.

Isolation of Murine Myeloid Progenitor Populations by CD34/CD150 Surface Markers

Myeloid progenitors are intermediates between Hematopoietic Stem Cells and their effector progeny. In mouse bone marrow, they are part of the Linage- Sca1- cKit+ (LK) compartment. Up to date, most researchers used CD34 and FcγR surface markers for dissection of this compartment into various populations. However, this approach does not provide distinct separation by fluorescence-activated cell sorting (FACS). In this study, we re-analyzed previously published single-cell index-sort and RNA-Seq. data and found that dissection of the LK compartment by surface expression of CD34 and CD150 provides better-defined populations of cells compared to the classical CD34/FcγR-based approach. We confirm our finding by FACS, and demonstrate the distinct differentiation potential of the newly-obtained LK sub-populations. Therefore, we suggest an alternative gating strategy for murine myeloid progenitors, utilizing commonly-used surface markers, and highlight the differences between populations based on transcriptome and phenotype.

Processed Baobab (Adansonia digitata L.) Food Products in Malawi: From Poor Men’s to Premium-Priced Specialty Food?

The baobab tree (Adansonia digitata L.) is an important source of non-timber forest products in sub-Saharan Africa. Its fruits contain high amounts of vitamin C, calcium, and dietary fibre. In addition, other parts of the tree are traditionally used for human consumption, particularly during lean seasons. In line with the increasing demand for natural, healthy, and nutritious food products, the baobab has great potential to contribute to human nutrition and rural livelihoods. In Malawi, where demand for baobab has substantially increased within the last decade, baobab fruits are being processed into a variety of food and non-food products, such as fruit juice, ice-lollies, sweets, and cosmetics. Yet, information on the sociodemographic background and quality preferences of baobab consumers is scanty. The current study, therefore, aimed to (1) map the diversity of baobab products available in Malawi; (2) determine consumer segments and their preferences for the most common baobab food products; and (3) examine the contribution of major attributes of processed baobab food products on their price. We employed a mixed-methods approach including the analysis of 132 baobab products and a survey of 141 consumers in formal and informal retail outlets, adopting multistage and purposive sampling. Qualitative and quantitative data were analysed using cluster analysis, cross tabulation, and hedonic regression. Results pointed to two distinct consumer segments for baobab food products, largely following the formal–informal product divide currently existing in Malawi. Both segments clearly differed with regard to preferred product attributes. We also showed that extrinsic product attributes such as packaging quality, labelling, conformity with food standards, or health claims provided distinct differentiation potential for baobab food manufacturers. In addition to providing empirical evidence for the transition of baobab food products into higher-value market segments, our results can help food processing enterprises to improve the composition and marketing of their baobab products.

Metabolic Phenotyping of Adipose-Derived Stem Cells Reveals a Unique Signature and Intrinsic Differences between Fat Pads.

White adipose tissues are functionally heterogeneous and differently manage the excess of energy supply. While the expansion of subcutaneous adipose tissues (SAT) is protective in obesity, that of visceral adipose tissues (VAT) correlates with the emergence of metabolic diseases. Maintained in fat pads throughout life, adipose stem cells (ASC) are mesenchymal-like stem cells with adipogenesis and multipotent differentiation potential. ASC from distinct fat pads have long been reported to present distinct proliferation and differentiation potentials that are maintained in culture, yet the origins of these intrinsic differences are still unknown. Metabolism is central to stem cell fate decision in line with environmental changes. In this study, we performed high-resolution nuclear magnetic resonance (NMR) metabolomic analyses of ASC culture supernatants in order to characterize their metabolic phenotype in culture. We identified and quantified 29 ASC exometabolites and evaluated their consumption or secretion over 72 h of cell culture. Both ASC used glycolysis and mitochondrial metabolism, as evidenced by the high secretions of lactate and citrate, respectively, but V-ASC mostly used glycolysis. By varying the composition of the cell culture medium, we showed that glutaminolysis, rather than glycolysis, supported the secretion of pyruvate, alanine, and citrate, evidencing a peculiar metabolism in ASC cells. The comparison of the two types of ASC in glutamine-free culture conditions also revealed the role of glutaminolysis in the limitation of pyruvate routing towards the lactate synthesis, in S-ASC but not in V-ASC. Altogether, our results suggest a difference between depots in the capacity of ASC mitochondria to assimilate pyruvate, with probable consequences on their differentiation potential in pathways requiring an increased mitochondrial activity. These results highlight a pivotal role of metabolic mechanisms in the discrimination between ASC and provide new perspectives in the understanding of their functional differences.

Multiple mesenchymal progenitor cell subtypes with distinct functional potential are present within the intimal layer of the hip synovium

BackgroundThe synovial membrane adjacent to the articular cartilage is home to synovial mesenchymal progenitor cell (sMPC) populations that have the ability to undergo chondrogenesis. While it has been hypothesized that multiple subtypes of stem and progenitor cells exist in vivo, there is little evidence supporting this hypothesis in human tissues. Furthermore, in most of the published literature on this topic, the cells are cultured before derivation of clonal populations. This gap in the literature makes it difficult to determine if there are distinct MPC subtypes in human synovial tissues, and if so, if these sMPCs express any markers in vivo/in situ that provide information in regards to the function of specific MPC subtypes (e.g. cells with increased chondrogenic capacity)? Therefore, the current study was undertaken to determine if any of the classical MPC cell surface markers provide insight into the differentiation capacity of sMPCs.MethodsClonal populations of sMPCs were derived from a cohort of patients with hip osteoarthritis (OA) and patients at high risk to develop OA using indexed cell sorting. Tri-differentiation potential and cell surface receptor expression of the resultant clones was determined.ResultsA number of clones with distinct differentiation potential were derived from this cohort, yet the most common cell surface marker profile on MPCs (in situ) that demonstrated chondrogenic potential was determined to be CD90+/CD44+/CD73+. A validation cohort was employed to isolate cells with only this cell surface profile. Isolating cells directly from human synovial tissue with these three markers alone, did not enrich for cells with chondrogenic capacity.ConclusionsTherefore, additional markers are required to further discriminate the heterogeneous subtypes of MPCs and identify sMPCs with functional properties that are believed to be advantageous for clinical application.

Single-cell analyses identify distinct and intermediate states of zebrafish pancreatic islet development.

Pancreatic endocrine islets are vital for glucose homeostasis. However, the islet developmental trajectory and its regulatory network are not well understood. To define the features of these specification and differentiation processes, we isolated individual islet cells from TgBAC(neurod1:EGFP) transgenic zebrafish and analyzed islet developmental dynamics across four different embryonic stages using a single-cell RNA-seq strategy. We identified proliferative endocrine progenitors, which could be further categorized by different cell cycle phases with the G1/S subpopulation displaying a distinct differentiation potential. We identified endocrine precursors, a heterogeneous intermediate-state population consisting of lineage-primed alpha, beta and delta cells that were characterized by the expression of lineage-specific transcription factors and relatively low expression of terminally differentiation markers. The terminally differentiated alpha, beta, and delta cells displayed stage-dependent differentiation states, which were related to their functional maturation. Our data unveiled distinct states, events and molecular features during the islet developmental transition, and provided resources to comprehensively understand the lineage hierarchy of islet development at the single-cell level.

Protein Expression Landscape Defines the Differentiation Potential Specificity of Adipogenic and Myogenic Precursors in the Skeletal Muscle.

The balance of adipogenic and myogenic differentiation of skeletal muscle-derived mesenchymal stem cells is particularly important in muscle development and intramuscular fat deposition. This study aimed to explore the differential regulation between adipogenic and myogenic precursors by comparative analysis of their global proteome expression profile. Adipogenic and myogenic precursors isolated from neonatal porcine longissimus dorsi muscle by the preplate method were verified for their unique and distinct differentiation potential under myogenic or adipogenic induction. A total of 433 differentially expressed proteins (DEP) ( P < 0.05 and FC > 1.20 or <0.83) between adipogenic and myogenic precursors were detected via a tandem mass tag (TMT)-coupled LC-MS/MS approach, including 339 up-regulated and 94 down-regulated proteins in myogenic precursors compared with adipogenic precursors. On the basis of functional annotation and enrichment analysis of 433 DEP, adipogenic and myogenic precursors showed significantly different metabolic pattern of energy substances and differential regulations of gene expression, cell structure and development, ion homeostasis, and cell motility and migration. Three pathways including PPAR signaling pathway, phosphatidylinositol signaling system, and autophagy signaling pathway, which was differentially regulated between adipogenic and myogenic precursors, was also discovered to play crucial roles in cell differentiation. In conclusion, these differentiated regulation patterns between the two cell subsets of mesenchymal precursors together defines their differentiation potential specificity.

Genetic and scRNA-seq Analysis Reveals Distinct Cell Populations that Contribute to Salivary Gland Development and Maintenance

Stem and progenitor cells of the submandibular salivary gland (SMG) give rise to, maintain, and regenerate the multiple lineages of mature epithelial cells including those belonging to the ductal, acinar, basal and myoepithelial subtypes. Here we have exploited single cell RNA-sequencing and in vivo genetic lineage tracing technologies to generate a detailed map of the cell fate trajectories and branch points of the basal and myoepithelial cell populations of the mouse SMG during embryonic development and in adults. Our studies show that the transcription factor p63 and alpha-smooth muscle actin (SMA) serve as faithful markers of the basal and myoepithelial cell lineages, respectively and that both cell types are endowed with progenitor cell properties. However, p63+ basal and SMA+ myoepithelial cells exhibit distinct cell fates by virtue of maintaining different cellular lineages during morphogenesis and in adults. Collectively, our results reveal the dynamic and complex nature of the diverse SMG cell populations and highlight the distinct differentiation potential of the p63 and SMA expressing subtypes in the stem and progenitor cell hierarchy. Long term these findings have profound implications towards a better understanding of the molecular mechanisms that dictate lineage commitment and differentiation programs during development and adult gland maintenance.