Key Stem Cell Regulators Research Articles

PBF-LB fabrication of microgrooves for induction of osteogenic differentiation of human mesenchymal stem cells

Stem cell differentiation has important implications for biomedical device design and tissue engineering. Recently, inherent material properties, including surface chemistry, stiffness, and topography, have been found to influence stem cell fate. Among these, surface topography is a key regulator of stem cells in contact with materials. The most important aspect of ideal bone tissue engineering is to control the organization of the bone extracellular matrix with fully differentiated osteoblasts. Here, we found that laser powder bed fusion (PBF-LB)-fabricated grooved surface inspired by the microstructure of bone, which induced human mesenchymal stem cell (hMSC) differentiation into the osteogenic lineage without any differentiation supplements. The periodic grooved structure was fabricated by PBF-LB which induced cell elongation facilitated by cytoskeletal tension along the grooves. This resulted in the upregulation of osteogenesis via Runx2 expression. The aligned hMSCs successfully differentiated into osteoblasts and further organized the bone mimetic-oriented extracellular matrix microstructure. Our results indicate that metal additive manufacturing technology has a great advantage in controlling stem cell fate into the osteogenic lineage, and in the construction of bone-mimetic microstructural organization. Our findings on material-induced stem cell differentiation under standard cell culture conditions open new avenues for the development of medical devices that realize the desired tissue regeneration mediated by regulated stem cell functions.

Single-Cell Multi-Omics Reveals the Genetic, Cellular and Molecular Landscape of TP53 Mutated Leukemic Transformation in MPN

Single-Cell Multi-Omics Reveals the Genetic, Cellular and Molecular Landscape of TP53 Mutated Leukemic Transformation in MPN

Conservation and diversification of HAIRY MERISTEM gene family in land plants.

The shoot apical meristems (SAMs) of land plants are crucial for plant growth and organ formation. In several angiosperms, the HAIRY MERISTEM (HAM) genes function as key regulators that control meristem development and stem cell homeostasis. To date, the origin and evolutionary history of the HAM family in land plants remains unclear. Potentially shared and divergent functions of HAM family members from angiosperms and non-angiosperms are also not known. In constructing a comprehensive phylogeny of the HAM family, we show that HAM proteins are widely present in land plants and that HAM proteins originated prior to the divergence of bryophytes. The HAM family was duplicated in a common ancestor of angiosperms, leading to two distinct groups: typeI and typeII. Type-II HAM members are widely present in angiosperms, whereas type-I HAM members were independently lost in different orders of monocots. Furthermore, HAM members from angiosperms and non-angiosperms (including bryophytes, lycophytes, ferns and gymnosperms) are able to replace the role of the type-II HAM genes in Arabidopsis, maintaining established SAMs and promoting the initiation of new stem cell niches. Our results uncover the conserved functions of HAM family members and reveal the conserved regulatory mechanisms underlying HAM expression patterning in meristems, providing insight into the evolution of key stem cell regulators in land plants.

3033 – SINGLE-CELL MULTI-OMIC ANALYSIS UNRAVELS GENETIC AND MOLECULAR HETEROGENEITY IN TRANSFORMED MYELOPROLIFERATIVE NEOPLASMS

One out of five patients with myeloproliferative neoplasms (MPN) will progress to an aggressive and largely untreatable form of Acute Myeloid Leukaemia (post-MPN secondary AML, or sAML). Transformation is associated with acquisition of TP53 missense mutations in approximately 50% of cases, and markedly increased genetic and molecular heterogeneity. This makes it challenging to effectively eliminate all subpopulations of tumour cells, and likely underlies the mostly universal treatment failure in sAML. Characterizing the complex genetic and cellular hierarchies associated with TP53 transformation is crucial for the development of effective targeted therapies. To resolve these multiple layers of intratumoral heterogeneity, we recently developed TARGET-seq (Rodriguez-Meira et al, 2019), a single-cell multi-omic method that correlates genetic, transcriptional and cell-surface proteomic readouts from the same single cell with extremely high resolution. TARGET-seq analysis of over 7000 Lineage-CD34+ cells (indexed for CD38/90/45RA/123) from TP53-mutant sAML patients revealed striking patterns of clonal evolution in different immunophenotypically defined stem/progenitor cell types, and identified preleukemic and leukemic subclones with markedly distinct molecular signatures. Acquisition of TP53 mutations was invariably associated with highly complex clonal evolution, enrichment of cell-cycle associated transcription and downregulation of key stem cell regulators. Interestingly, preleukemic subclones from patients undergoing transformation were highly enriched in WNT-beta catenin signaling and depleted in DNA-repair genes, as compared to non-transformed MPN patients. This suggests that preleukemic clones could be primed for disease transformation, and could potentially be targeted to prevent disease progression. Ultimately, single-cell multi-omic analysis identified key molecular regulators of TP53 mediated transformation, providing unique insights into the evolution of these tumors and how they might be therapeutically targeted.

Sexual dimorphism of niche architecture and regulation of the Caenorhabditis elegans germline stem cell pool.

Stem cell maintenance by niche signaling is a common theme across phylogeny. In the Caenorhabditis elegans gonad, the broad outlines of germline stem cell (GSC) regulation are the same for both sexes: GLP-1/Notch signaling from the mesenchymal distal tip cell niche maintains GSCs in the distal gonad of both sexes and does so via two key stem cell regulators, SYGL-1 and LST-1. Yet most recent analyses of niche signaling and GSC regulation have focused on XX hermaphrodites, an essentially female sex making sperm in larvae and oocytes in adults. Here we focus on GSC regulation in XO males. Sexual dimorphism of niche architecture, reported previously, suggested that the molecular responses to niche signaling or numbers of GSCs might also be sexually distinct. Remarkably, this is not the case. This work extends our understanding of the sexually dimorphic niche architecture, but also demonstrates that the dimorphic niches drive a similar molecular response and maintain a similar number of GSCs in their stem cell pools.

Sumoylation of Notch1 represses its target gene expression during cell stress

The Notch signaling pathway is a key regulator of stem cells during development, and its deregulated activity is linked to developmental defects and cancer. Transcriptional activation of Notch target genes requires cleavage of the Notch receptor in response to ligand binding, production of the Notch intracellular domain (NICD1), NICD1 migration into the nucleus, and assembly of a transcriptional complex. Post-translational modifications of Notch regulate its trafficking, turnover, and transcriptional activity. Here, we show that NICD1 is modified by small ubiquitin-like modifier (SUMO) in a stress-inducible manner. Sumoylation occurs in the nucleus where NICD1 is sumoylated in the RBPJ-associated molecule (RAM) domain. Although stress and sumoylation enhance nuclear localization of NICD1, its transcriptional activity is attenuated. Molecular modeling indicates that sumoylation can occur within the DNA-bound ternary transcriptional complex, consisting of NICD1, the transcription factor Suppressor of Hairless (CSL), and the co-activator Mastermind-like (MAML) without its disruption. Mechanistically, sumoylation of NICD1 facilitates the recruitment of histone deacetylase 4 (HDAC4) to the Notch transcriptional complex to suppress Notch target gene expression. Stress-induced sumoylation decreases the NICD1-mediated induction of Notch target genes, which was abrogated by expressing a sumoylation-defected mutant in cells and in the developing central nervous system of the chick in vivo. Our findings of the stress-inducible sumoylation of NICD1 reveal a novel context-dependent regulatory mechanism of Notch target gene expression.

Sirtinol, a Sir2 protein inhibitor, affects stem cell maintenance and root development in Arabidopsis thaliana by modulating auxin-cytokinin signaling components

In Arabidopsis thaliana, besides several key transcription factors and chromatin modifiers, phytohormones auxin and cytokinin play pivotal role in shoot and root meristem maintenance, and lateral root (LR) development. Sirtinol, a chemical inhibitor of Sir2 proteins, is known to promote some auxin induced phenotypes in Arabidopsis. However, its effect on plant stem cell maintenance or organ formation remained unaddressed. Here we show that sirtinol affects meristem maintenance by altering the expression of key stem cell regulators, cell division and differentiation by modulating both auxin and cytokinin signaling in Arabidopsis thaliana. The expression of shoot stem cell niche related genes WUSCHEL (WUS) and CLAVATA3 (CLV3) was upregulated, whereas SHOOT MERISTEMLESS (STM) was downregulated in sirtinol treated seedlings. The expression level and domain of key root stem cell regulators PLETHORA (PLTs) and WUS-Related Homeobox 5 (WOX5) were altered in sirtinol treated roots. Sirtinol affects LR development by disturbing proper auxin transport and maxima formation, similar to 2,4-dichlorophenoxyacetic acid (2,4-D). Sirtinol also affects LR formation by altering cytokinin biosynthesis and signaling genes in roots. Therefore, sirtinol affects shoot and root growth, meristem maintenance and LR development by altering the expression of cytokinin-auxin signaling components, and regulators of stem cells, meristems, and LRs.

Abstract B21: DNp63 promotes stem cell activity in mammary gland development and basal-like breast cancer by enhancing Fzd7 expression and Wnt signaling

Abstract Emerging evidence suggests that cancer is populated and maintained by tumor initiating cells (TICs) with stem-like properties similar to that of adult tissue stem cells. Despite recent advances, the molecular regulatory mechanisms that may be shared between normal and malignant stem cells remain poorly understood. Here we show that the DNp63 isoform of the Trp63 transcription factor promotes normal mammary stem cell (MaSC) activity by increasing the expression of the Wnt receptor Fzd7 and thereby enhancing Wnt signaling. Importantly, Fzd7-dependent enhancement of Wnt signaling by DNp63 also governs tumor initiating activity of the basal subtype of breast cancer. These findings establish DNp63 as a key regulator of stem cells in both normal and malignant mammary tissues and provide direct evidence that breast cancer TICs and normal MaSCs share common regulatory mechanisms. Citation Format: Rumela Chakrabarti, Satrajit Sinha, Yibin Kang. DNp63 promotes stem cell activity in mammary gland development and basal-like breast cancer by enhancing Fzd7 expression and Wnt signaling. [abstract]. In: Proceedings of the AACR Special Conference: Developmental Biology and Cancer; Nov 30-Dec 3, 2015; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(4_Suppl):Abstract nr B21.

Preferential Propagation of Competent SIX2+ Nephronic Progenitors by LIF/ROCKi Treatment of the Metanephric Mesenchyme

SummaryUnderstanding the mechanisms responsible for nephrogenic stem cell preservation and commitment is fundamental to harnessing the potential of the metanephric mesenchyme (MM) for nephron regeneration. Accordingly, we established a culture model that preferentially expands the MM SIX2+ progenitor pool using leukemia inhibitory factor (LIF), a Rho kinase inhibitor (ROCKi), and extracellular matrix. Passaged MM cells express the key stem cell regulators Six2 and Pax2 and remain competent to respond to WNT4 induction and form mature tubular epithelia and glomeruli. Mechanistically, LIF activates STAT, which binds to a Stat consensus sequence in the Six2 proximal promoter and sustains SIX2 levels. ROCKi, on the other hand, attenuates the LIF-induced differentiation activity of JNK. Concomitantly, the combination of LIF/ROCKi upregulates Slug expression and activates YAP, which maintains SIX2, PAX2, and SALL1. Using this novel model, our study underscores the pivotal roles of SIX2 and YAP in MM stem cell stability.

Reactive oxygen species-mediated therapeutic response and resistance in glioblastoma.

Glioblastoma (GBM) resistance to therapy is the most common cause of tumor recurrence, which is ultimately fatal in 90% of the patients 5 years after initial diagnosis. A sub-population of tumor cells with stem-like properties, glioma stem cells (GSCs), is specifically endowed to resist or adapt to the standard therapies, leading to therapeutic resistance. Several anticancer agents, collectively termed redox therapeutics, act by increasing intracellular levels of reactive oxygen species (ROS). In this study, we investigated mechanisms underlying GSC response and resistance to cannabidiol (CBD), a non-toxic, non-psychoactive cannabinoid and redox modulator. Using primary GSCs, we showed that CBD induced a robust increase in ROS, which led to the inhibition of cell survival, phosphorylated (p)-AKT, self-renewal and a significant increase in the survival of GSC-bearing mice. Inhibition of self-renewal was mediated by the activation of the p-p38 pathway and downregulation of key stem cell regulators Sox2, Id1 and p-STAT3. Following CBD treatment, a subset of GSC successfully adapted, leading to tumor regrowth. Microarray, Taqman and functional assays revealed that therapeutic resistance was mediated by enhanced expression of the antioxidant response system Xc catalytic subunit xCT (SLC7A11 (solute carrier family 7 (anionic amino-acid transporter light chain), member 11)) and ROS-dependent upregulation of mesenchymal (MES) markers with concomitant downregulation of proneural (PN) markers, also known as PN–MES transition. This ‘reprogramming' of GSCs occurred in culture and in vivo and was partially due to activation of the NFE2L2 (NRF2 (nuclear factor, erythroid 2-like)) transcriptional network. Using genetic knockdown and pharmacological inhibitors of SLC7A11, we demonstrated that combining CBD treatment with the inhibition of system Xc resulted in synergistic ROS increase leading to robust antitumor effects, that is, decreased GSC survival, self-renewal, and invasion. Our investigation provides novel mechanistic insights into the antitumor activity of redox therapeutics and suggests that combinatorial approaches using small molecule modulators of ROS offer therapeutic benefits in GBM.

ΔNp63 promotes stem cell activity in mammary gland development and basal-like breast cancer by enhancing Fzd7 expression and Wnt signalling.

Emerging evidence suggests that cancer is populated and maintained by tumor initiating cells (TICs) with stem-like properties similar to that of adult tissue stem cells. Despite recent advances, the molecular regulatory mechanisms that may be shared between normal and malignant stem cells remain poorly understood. Here we show that the ΔNp63 isoform of the Trp63 transcription factor promotes normal mammary stem cell (MaSC) activity by increasing the expression of the Wnt receptor Fzd7, thereby enhancing Wnt signaling. Importantly, Fzd7-dependent enhancement of Wnt signaling by ΔNp63 also governs tumor initiating activity of the basal subtype of breast cancer. These findings establish ΔNp63 as a key regulator of stem cells in both normal and malignant mammary tissues and provide direct evidence that breast cancer TICs and normal MaSCs share common regulatory mechanisms.

Angiopep-2-conjugated liposomes encapsulating γ-secretase inhibitor for targeting glioblastoma stem cells

CD133+ cell subpopulation in U87 MG cells displays glioblastoma stem cell (GSC) like properties. Notch, a key regulator of stem cells, is also over-activated in GSCs. It was previously reported that γ-secretase inhibitors inhibit the Notch pathway by targeting the γ-secretase complex. To examine the GSCs, CD133+ cells were separated from the U87 MG cells by magnetic activated cell sorting and only cell populations with nearly 90 % CD133+ cells were used as GSCs. The sorted U87-CD133+ cells indeed display enhanced chemoresistance and high Notch activity. For targeting studies, Angiopep-2 (An2)-conjugated YO-01027 (YO) encapsulating liposomes (PEG-lipo-YO-An2) were prepared by lipid film hydration method. An2 is an effective ligand of low density lipoprotein receptor-related protein which is over-expressed in the blood–brain barrier and GSCs. The mean diameter, zeta potential and encapsulation efficiency of PEG-lipo-YO-An2 was around 180 nm, −10.0 mV and 56.6 %, respectively. In in vitro studies, we confirmed that PEG-lipo-YO-An2 showed enhanced anti-GSC properties such as enhanced stability, anti-proliferation and anti-tumor sphere formation abilities towards than free drug. This study demonstrates that An2 conjugation and liposomal encapsulation of YO enhance the cytotoxicity of YO against GSCs, and this formulation could be used as a promising candidate for the treatment of glioblastoma multiforme by targeting GSCs.

Mesoporous Silica Nanoparticles as Drug Delivery Systems for Targeted Inhibition of Notch Signaling in Cancer

Notch signaling, a key regulator of stem cells, is frequently overactivated in cancer. It is often linked to aggressive forms of cancer, evading standard treatment highlighting Notch as an exciting therapeutic target. Notch is in principle "druggable" by γ-secretase inhibitors (GSIs), inhibitory peptides and antibodies, but clinical use of Notch inhibitors is restricted by severe side effects and there is a demand for alternative cancer-targeted therapy. Here, we present a novel approach, using imagable mesoporous silica nanoparticles (MSNPs) as vehicles for targeted delivery of GSIs to block Notch signaling. Drug-loaded particles conjugated to targeting ligands induced cell-specific inhibition of Notch activity in vitro and exhibited enhanced tumor retainment with significantly improved Notch inhibition and therapeutic outcome in vivo. Oral administration of GSI-MSNPs controlled Notch activity in intestinal stem cells further supporting the in vivo applicability of MSNPs for GSI delivery. MSNPs showed tumor accumulation and targeting after systemic administration. MSNPs were biocompatible, and particles not retained within the tumors, were degraded and eliminated mainly by renal excretion. The data highlights MSNPs as an attractive platform for targeted drug delivery of anticancer drugs with otherwise restricted clinical application, and as interesting constituents in the quest for more refined Notch therapies.

Heme oxygenase-1 deficiency leads to disrupted response to acute stress in stem cells and progenitors

AbstractAn effective response to extreme hematopoietic stress requires an extreme elevation in hematopoiesis and preservation of hematopoietic stem cells (HSCs). These diametrically opposed processes are likely to be regulated by genes that mediate cellular adaptation to physiologic stress. Herein, we show that heme oxygenase-1 (HO-1), the inducible isozyme of heme degradation, is a key regulator of these processes. Mice lacking one allele of HO-1 (HO-1+/−) showed accelerated hematopoietic recovery from myelotoxic injury, and HO-1+/− HSCs repopulated lethally irradiated recipients with more rapid kinetics. However, HO-1+/− HSCs were ineffective in radioprotection and serial repopulation of myeloablated recipients. Perturbations in key stem cell regulators were observed in HO-1+/− HSCs and hematopoietic progenitors (HPCs), which may explain the disrupted response of HO-1+/− HPCs and HPCs to acute stress. Control of stem cell stress response by HO-1 presents opportunities for metabolic manipulation of stem cell–based therapies.