Primitive Stem Cell Population Research Articles

A Rapid Test to Evaluate the Tumor Differentiation State of Acute Myeloid Leukemias

Introduction: The combination of the hypomethylating agent Azacitidine with Venetoclax, a BCL2 inhibitor (Aza+Ven), is the frontline therapy for patients with acute myeloid leukemia (AML) who are elderly or unfit for intensive chemotherapy. However, identifying responsiveness to Aza+Ven a priori remains a clinical challenge.Aza+Ven effectively targets the primitive leukemia stem cell population (Pollyea, 2018), whereas resistance is observed in differentiated tumors (Pei, 2020). Further insights into the hierarchical organization of hematopoiesis have led to a definition of malignant AML cell types that follow the progression from hematopoietic stem cell to myeloid differentiated cells based on gene expression signatures, and these have been used to classify AML tumors (van Galen, 2019). Classification schemes using gene expression to capture variations in leukemic composition represent key biomarkers of response to targeted agents (Zeng, 2022; Bottomly, 2022). However, the prompt availability of nuanced cell state signatures to guide clinical decision making is limited by the laborious nature of performing and analyzing RNA-sequencing studies. Rapid AML stratification based on these classifications permits the possibility of identifying patients most likely to benefit from a drug or combination. Methods: To classify cell states based on transcriptomic signatures, we developed two assay formats, involving custom Nanostring and TaqMan Array card panels, for evaluation of tumor cell states for AML patients. Both formats contained gene sets representative of the six hematopoietic lineage states (van Galen, 2019) and both provide outputs that are compatible with the timeline for clinical decision-making. Assays were run according to the manufacturers' specifications, and enrichment of each of the six gene signatures was assessed using the Singscore stable method (Foroutan, 2018). Banked AML patient samples with matched RNA-seq and ex vivo Aza+Ven drug sensitivity data were used to validate the assay formats. Data obtained from the rapid assays were validated against cell states determined by deconvolution of bulk RNA sequence and compared with respect to clinical immunophenotype data where available. Results : Both the Nanostring and quantitative PCR-based assay formats detected gene expression representative of the appropriate cell state as previously determined by bulk RNA sequencing (Fig. 1A). Correlation of Aza+Ven ex vivo sensitivity with tumor cell state signatures revealed patterns where primitive but not differentiated cell states associated with sensitivity. In contrast, RNA isolated from healthy donor samples showed expression of all six cell state signatures. Across a cohort of 50 primary AML patient specimens, enrichment scores for each gene set correlated well with those derived for the same patients using deconvolution of bulk RNA-seq (Pearson r values: 0.785 to 0.944). Cell state characterization by rapid gene expression assays also aligned well with clinical immunophenotype, a routinely available diagnostic characterization of tumor blasts. Surface expression of primitive (CD117) and mature (CD11b) markers associated with Aza+Ven ex vivo sensitivity and resistance overall, respectively (p=0.03 & 0.04); however, ex vivo sensitivity could be significantly further stratified among samples expressing these markers when considering the gene expression-based enrichment scores for progenitor- and monocyte-like gene signatures (low vs high p-values: 0.02-0.05) (Fig. 1B). Analysis of matched samples from individual patients before and after treatment also showed cell state signature changes that reflected their temporal disease progression and correlated with differences in ex vivo drug sensitivity and clinical immunophenotype. Conclusion:Incorporation of cell state into prospective evaluations of AML patients provides an early indication of responsiveness to Aza+Ven. With rapid turnaround, assays such as these may augment treatment stratification and improve patient outcomes. These assays offer additional versatility to detect changes in AML cells after forced differentiation or acquired drug resistance.

De-osteogenic-differentiated mesenchymal stem cells accelerate fracture healing by mir-92b.

BackgroundMesenchymal stem cells (MSCs) are promising targets for therapeutic use in regenerative medicine and tissue engineering. In the previous study, we have found that MSCs could be reverted to a primitive stem cell population after in vitro induction of osteogenic and de-osteogenic differentiation (de-osteogenic differentiated MSCs, De-Os-MSCs). De-Os-MSCs showed improved cell survival and osteogenic potential. However, the underlying mechanism and its potential effect on fracture healing has not been explored.MethodsMSCs were isolated from the rat bone marrow. MicroRNAs were cloned into lentiviral vectors and transduced into MSCs to observe the effects on osteogenesis. The expression levels of marker genes were evaluated by quantitative RT-PCR. Ectopic bone formation model was used to evaluate the bone regeneration ability of mir-92b transduced MSCs in vivo. An open femur fracture model was established, and MSCs or De-Os-MSCs were administrated to the fracture sites. Histological, biomechanical and microCT analysis were used to evaluate the quality of bone.ResultsIn the present study, we found that mir-92b was significantly increased in the secretions of De-Os-MSCs. And mir-92b could promote the osteogenic differentiation potential of MSCs by activating pERK and JNK signaling pathways. The ectopic bone formation assay showed that MSCs overexpressing mir-92b formed more bone like tissues in vivo. Most importantly, we found local administration of De-Os-MSCs could accelerate fracture healing using an open femur fracture model in rats. The quality of bone property was much better as shown by microCT and biomechanical testing.ConclusionTaken together, our study demonstrated that mir-92b promoted osteogenesis of MSCs, which was partially accounted for the enhanced osteogenic differentiation potential of De-Os-MSCs. And De-Os-MSCs had shown better regenerative capacity in accelerating fracture healing when they were locally given.The translational potential of this articleDe-Os-MSCs could be used to accelerate fracture healing, and reduce the occurrence of delayed unions and non-unions.

Rigosertib in Combination with Azacitidine Impacts Metabolic and Differentiation Pathways in the MDS-L Cell Line

Background Myelodysplastic Syndrome (MDS) is characterized by ineffective clonal hematopoiesis with peripheral blood cytopenias, leading to death from infection or bleeding. Azacitidine (AZA), a hypomethylating agent (HMA) is the standard of care for treatment of MDS patients (pts) with higher-risk MDS [Silverman LR, The Myelodysplastic Syndrome in Cancer Medicine, Editors: R.J. Bast, et al. 2017]. Responses to AZA occur in 50% of pts with significant effects on hematopoiesis ranging from improvement in a single lineage to complete restoration of blood counts and transfusion independence [Silverman LR, et al. Leukemia, 1993]. AZA treatment is associated with global DNA hypomethylation, including human endogenous retroviruses (HEV) which further activates innate immune signaling [Chiappinelli KB,et al. Cell, 2015]. The exact mechanism by which AZA improves hematopoiesis is unknown. AZA improves overall survival of pts, yet despite this, 100% of pts ultimately fail treatment with worsening cytopenias or transformation to leukemia [Silverman LR, et al. B. J Clin Oncol, 2002; Cancer, 2011]. Thus, understanding the mechanism of resistance and identification of targets which can reverse HMA failure and improve hematopoiesis in MDS pts is critical. Our clinical data demonstrate that AZA combined with Rigosertib (RIGO), a novel Ras mimetic that inhibits Ras/Raf signaling [Athuluri-Divakar SK, et al. Cell, 2016], yields a response rate of 54% of pts who were HMA failures [Navada SC, et al. EHA 2019]. The response was associated with significant improvement in hematopoiesis and represents a critical observation in overcoming the epigenetic clinical resistance phenotype. The precise mechanism that leads to reversal of the resistance phenotype is poorly understood. Methods: We investigated the differential protein expression in response to different treatment (AZA, RIGO alone and sequential combinations RIGO/AZA; AZA/RIGO) in vitro in MDS-L cell line by Reverse phase protein array (RPPA). Further, we also studied the functional role of these treatments on differentiation in the cell line by growing cells on semi-solid media as well as by flow cytometry using various stem cell and differentiation markers (CD34, CD38, CD45, CD123). Results: RPPA analysis indicated a discrete responses to treatment in the MDS-L cell line. The response was prominent with differential expression of 43 proteins specifically in MDS-L cells treated with RIGO/AZA that include PIK3R1, AKT1, mTOR, p38 MAPK, PTEN, RPS6KA1 (Fold change (FC) < -2). All of these proteins are downregulated which suggests inhibition of PI3K and mTOR signaling. Proteins belonging to metabolic pathways, including ACC1 and ACLY, were found to be downregulated, whereas proteins related to mitochondrial function and oxidative phosphorylation (OXPHOS) were upregulated (FC > 1.5/< -1.5) in cells treated with RIGO/AZA. In addition, we found that MDS-L cells represented both CD34+CD38+ and CD34+CD38- populations by FACS analysis (Fig 1). AZA increases the percentage of CD34+CD38+, indicative of differentiation, whereas RIGO alone increased the percentage of CD34+CD38- cells, representing a primitive stem cell population (Table in Fig 1). Based on the combination of differentiation markers (illustrated in fig 1), we observed that RIGO alone, and sequenced as combination RIGO/AZA, impacts different progenitors such as granulocyte-macrophage progenitor (GMP), megakaryocyte erythroid progenitor (MEP), and multipotent progenitor (MPP). RIGO/AZA treatment showed a decrease in GMP, while the % of MEP was increased, as compared to other treatments. Moreover, we found a remarkable reduction in the number of colony forming units on differentiation media in response to RIGO (83%) and RIGO/AZA (90%). Conclusions: These data demonstrate that in addition to inhibition of the PI3/AKT/mTOR pathway, the RIGO/AZA combination also impacts metabolic and differentiation pathways of MDS-L cells. RIGO alone appears to promote maintenance of a primitive stem cell population, while the RIGO/AZA sequenced combination appears to push the cells toward a cycling stage with increased expression of genes associated with OXPHOS. In comparison, when treated with RIGO, cells remain in a less differentiated stage. Further studies are underway to determine the effect of metabolic changes on differentiation and maintenance of hematopoietic stem cells. Figure Disclosures Navada: Onconova Therapeutics Inc: Research Funding. Reddy:Onconova Therapeutics Inc: Research Funding. Silverman:Celgene: Research Funding; Medimmune: Research Funding; Onconova Therapeutics Inc: Patents & Royalties, Research Funding.

Renogenic characterization and in vitro differentiation of rat amniotic fluid stem cells into renal proximal tubular- and juxtaglomerular-like cells.

The aim of the present study was to investigate the renogenic characteristics of amniotic fluid stem cells (AFSCs) and to evaluate their in vitro differentiation potential into renal proximal tubular-like cells and juxtaglomerular-like cells. We culture expanded AFSCs derived from rat amniotic fluid. The AFSCs grew as adherent spindle-shaped cells and expressed mesenchymal markers CD73, CD90, and CD105 as well as renal progenitor markers WT1, PAX2, SIX2, SALL1, and CITED1. AFSCs exhibited an in vitro differentiation potential into renal proximal tubular epithelial-like cells, as shown by the upregulation of expression of proximal tubular cell-specific genes like AQP1, CD13, PEPT1, GLUT5, OAT1, and OCT1. AFSCs could also be differentiated into juxtaglomerular-like cells as demonstrated by the expression of renin and α-SMA. The AFSCs also expressed pluripotency markers OCT4, NANOG, and SOX2 and could be induced into embryoid bodies with differentiation into all the three germ layers, highlighting the pluripotent nature of these cells. Our results show that amniotic fluid contains a population of primitive stem cells that express renal-progenitor markers and also possess the propensity to differentiate into two renal lineage cell types and, thus, may have a therapeutic potential in renal regenerative medicine.

Polyphenols Modulate Quiescence/Mobilization of Hematopoietic Immature Cells through APC/EPCR/PAR-1 Axis

Coordinated arrangement of bone marrow (BM) microenvironment is essential for the maintenance of hematopoietic stem cells and their progenitors (HSPC), and for the continuous supply of peripheral blood (PB) cells. Quiescence and/or mobilization of these BM cells into the circulation are tightly regulated however non-physiological or stress conditions, such as infections, can accelerate them. Our previous findings have shown that polyphenols from green tea modulate the myeloid population (Gr-1+Mac-1+) of BM, spleen and PB of mice challenged by lipopolysaccharide (LPS). We have also observed a reduction in the circulating levels of thrombin-antithrombin complex (TAT) and soluble endothelial protein C receptor (sEPCR) of these mice. It has been reported that thrombin induces the rapid HSPC mobilization through coagulation thrombin/PAR-1 axis, and quiescence is maintained across the APC/EPCR/PAR-1 axis (Nat. Med. 2015, 21:1307-17). In this context, our goal was to investigate the effects of polyphenols on the Lin-Sca-1+c-Kit+ (LSK) primitive stem cell population and mature populations including: T and B lymphocytes, monocytes, granulocytes and erythrocytes (Lin+). The expression of PAR-1 and EPCR in these cells, and the levels of sEPCR in the BM fluid were also evaluated. The 250 mg/kg dose of polyphenol from green tea extract was given once every 7 days orally (gavage) to mice (n=6/group) challenged with i.p. injection of 100µg LPS. Control group received vehicle only. After 24h, mice were sacrificed; BM and PB were collected for the assays. LPS injection induced an increase in circulating LSK, without affecting the pool of these cells in BM. Treatment with polyphenols prevented recruitment of LSK into the PB, and did not alter the percentage of BM LSK. Corroborating these findings, polyphenols also reduced the number of immature progenitors (CFU-C) in the PB, evaluated by the clonal culture assay. In addition, polyphenols did not affect the increase in the number of Lin+ cells and circulating leukocytes (total WBC) induced by LPS injection. We also observed that LPS induced a rapid increase in the number of PB LSK and Lin+ cells expressing PAR-1, while reducing BM LSK expressing EPCR. Polyphenols did not affect PAR-1 expression in both populations, but partially recovered expression of EPCR in BM LSK. Polyphenols also reduced the increased levels of sEPCR induced by LPS in the BM niche. Taken together, our results demonstrate that polyphenols promote an effect on the quiescence/mobilization of both the most primitive hematopoietic cells and their progenitors, but does not affect mature populations. The reduction in vascular permeability observed in the BM of mice using Evans blue reinforces this action of polyphenols. It appears that the effect of polyphenols is thrombin dependent; although polyphenols did not affect the expression of PAR-1, they modulated EPCR, mainly in the medullary microenvironment with maintenance of barrier integrity and consequent cellular quiescence. In addition, polyphenols appear to have an anti-inflammatory effect, possibly triggered by their effect on the EPCR. DisclosuresDe Paula:Hematology and Transfusion Medicine Center, University of Campinas: Employment. Shiraishi:Hematology and Transfusion Medicine Center, University of Campinas: Employment. Queiroz:University of Campinas: Employment.

SMALL Molecules Mediated Hematopoietic STEM and Progenitor CELLS Expansion for GENE Editing Application

Genome editing of Hematopoietic stem Cells has revolutionized the treatment strategies for genetic disorders. Despite this, it still remains a great challenge as hematopoietic stem cells tend to lose its stem-ness during the ex vivo culture and gene editing process. The need for large dose of CD34+ HSPCs for manipulation makes it a seemingly difficult strategy. Recent works suggest that the potential effects of small molecules in expanding cord blood HSPCs ex vivo promoting self-renewal and delaying differentiation. We screened several reported small molecules to identify a condition that promotes the expansion of adult HSPCs for gene manipulation process.The mobilized Peripheral blood HSPCs are purified and cultured with a cytokine cocktail. Along with the cytokine cocktail, we tested several small molecules and in different combinations. Expression of cell surface receptors were analysed by FACS after 12 days of ex vivo culture. Our screening identified a unique culture condition that expanded the primitive stem cell population (CD34+/CD133+/CD90+cells) along with the early progenitors (CD34+/CD133+) and the progenitors (CD34+).Our culture conditions expanded the primitive cells by 20 folds compared to the mock treated cells. Our treatment release experiments suggested that the expansion is due to our culture conditions and are reversible.The colony forming cell (CFC) assay showed about 30 fold increase in the numbers of multilineage colony forming cell (CFU-GEMM) thereby ensuring the proliferation and differentiation capacity of expanded HSPCs. Their differentiation ability was also confirmed by ex vivo differentiation into Megakaryocytes. Our treatment conditions reduced the apoptosis rate during the ex vivo culture and improved their cell migration response towards SDF. The reduced reactive oxygen species levels and increased CXCR4 expression were observed in our expanded HSPCs and these might be the possible reasons for the low apoptosis and better cell migration respectively. DisclosuresNo relevant conflicts of interest to declare.

Dynamics of genetically engineered hematopoietic stem and progenitor cells after autologous transplantation in humans.

Hematopoietic stem and progenitor cells (HSPC) are endowed with the role of generating and maintaining lifelong the extremely diverse pool of blood cells1. Clinically, transplantation of human HSPC from an allogeneic healthy donor or infusion of autologous gene-corrected HSPC can effectively replenish defective blood cell production caused by congenital or acquired disorders2-9. However, due to methodological and ethical constraints that have limited the study of human HSPC primarily to in vitro assays10 or xenotransplantation models11,12, the in vivo activity of HSPC has to date remained relatively unexplored in humans13-16. Here we report a comprehensive study of the frequencies, dynamics and output of seven HSPC subtypes in humans that was performed by tracking 148,093 individual clones in six patients treated with lentiviral gene therapy using autologous HSPC transplantation and followed for up to 5 years. We discovered that primitive multipotent progenitor and hematopoietic stem cell (HSC) populations have distinct roles during the initial reconstitution after transplant, compared with subsequent steady-state phases. Furthermore, we showed that a fraction of in vitro-activated HSC are resilient and undergo a defined delayed activation period upon transplant. Finally, our data support the concept that early lymphoid-biased progenitors might be capable of long-term survival, such that they can be maintained independently of their continuous production from HSC. Overall, this study provides comprehensive data on HSPC dynamics after autologous transplantation and gene therapy in humans.

Cobll1 is linked to drug resistance and blastic transformation in chronic myeloid leukemia.

Drug resistance to BCR-ABL1 tyrosine kinase inhibitor (TKI) and disease progression to blast crisis (BC) are major clinical problems in chronic myeloid leukemia (CML); however, underlying mechanisms governing this process remain to be elucidated. Here, we report Cordon-bleu protein-like 1 (Cobll1) as a distinct molecular marker associated with drug resistance as well as progression to BC. In detail, Cobll1 increases IKKγ stability, leading to NF-κB activation and reduction of nilotinib-dependent apoptosis, suggesting Cobll1-mediated NF-κB could be involved in drug resistance. Recently, NF-κB signalling has been highlighted as a core mechanism for chronic phase (CP)-BC progression, stem cell survival and tyrosine kinase inhibitor resistance. We also demonstrated that high expression of Cobll1 confers drug resistance to tyrosine kinase inhibitors in CML cell line as well as patient samples. The analysis of large sets of primary CML samples (n=90) shows that Cobll1 expression is dramatically increased in BC but not in CP, which is correlated with a poor survival rate (P=0.002). Moreover, our studies show that Cobll1 is highly expressed in CD34+ primitive stem cell populations, and the zebrafish paralog Cobll1b is important for normal hematopoiesis during embryonic development. Based on these results, we propose that Cobll1 is a novel biomarker and potential therapeutic target for CML-BC.

Epigenetic Modification of the CCL5/CCR1/ERK Axis Enhances Glioma Targeting in Dedifferentiation-Reprogrammed BMSCs

SummaryThe success of stem cell-mediated gene therapy in cancer treatment largely depends on the specific homing ability of stem cells. We have previously demonstrated that after in vitro induction of neuronal differentiation and dedifferentiation, bone marrow stromal cells (BMSCs) revert to a primitive stem cell population (De-neu-BMSCs) distinct from naive BMSCs. We report here that De-neu-BMSCs express significantly higher levels of chemokines, and display enhanced homing abilities to glioma, the effect of which is mediated by the activated CCL5/CCR1/ERK axis. Intriguingly, we find that the activated chemokine axis in De-neu-BMSCs is epigenetically regulated by histone modifications. On the therapeutic front, we show that De-neu-BMSCs elicit stronger homing and glioma-killing effects together with cytosine deaminase/5-fluorocytosine compared with unmanipulated BMSCs in vivo. Altogether, the current study provides an insight into chemokine regulation in BMSCs, which may have more profound effects on BMSC function and their application in regenerative medicine and cancer targeting.

A label-retaining but unipotent cell population resides in biliary compartment of mammalian liver

Cells with slow proliferation kinetics that retain the nuclear label over long time periods–the label-retaining cells (LRCs)–represent multipotent stem cells in a number of adult tissues. Since the identity of liver LRCs (LLRCs) had remained elusive we utilized a genetic approach to reveal LLRCs in normal non-injured livers and characterized their regenerative properties in vivo and in culture. We found that LLRCs were located in biliary vessels and participated in the regeneration of biliary but not hepatocyte injury. In culture experiments the sorted LLRCs displayed an enhanced self-renewal capacity but a unipotent biliary differentiation potential. Transcriptome analysis revealed a unique set of tumorigenesis- and nervous system-related genes upregulated in LLRCs when compared to non-LRC cholangiocytes. We conclude that the LLRCs established during the normal morphogenesis of the liver do not represent a multipotent primitive somatic stem cell population but act as unipotent biliary progenitor cells.

ICRP Publication 131: Stem Cell Biology with Respect to Carcinogenesis Aspects of Radiological Protection.

This report provides a review of stem cells/progenitor cells and their responses to ionising radiation in relation to issues relevant to stochastic effects of radiation that form a major part of the International Commission on Radiological Protection's system of radiological protection. Current information on stem cell characteristics, maintenance and renewal, evolution with age, location in stem cell 'niches', and radiosensitivity to acute and protracted exposures is presented in a series of substantial reviews as annexes concerning haematopoietic tissue, mammary gland, thyroid, digestive tract, lung, skin, and bone. This foundation of knowledge of stem cells is used in the main text of the report to provide a biological insight into issues such as the linear-no-threshold (LNT) model, cancer risk among tissues, dose-rate effects, and changes in the risk of radiation carcinogenesis by age at exposure and attained age. Knowledge of the biology and associated radiation biology of stem cells and progenitor cells is more developed in tissues that renew fairly rapidly, such as haematopoietic tissue, intestinal mucosa, and epidermis, although all the tissues considered here possess stem cell populations. Important features of stem cell maintenance, renewal, and response are the microenvironmental signals operating in the niche residence, for which a well-defined spatial location has been identified in some tissues. The identity of the target cell for carcinogenesis continues to point to the more primitive stem cell population that is mostly quiescent, and hence able to accumulate the protracted sequence of mutations necessary to result in malignancy. In addition, there is some potential for daughter progenitor cells to be target cells in particular cases, such as in haematopoietic tissue and in skin. Several biological processes could contribute to protecting stem cells from mutation accumulation: (a) accurate DNA repair; (b) rapidly induced death of injured stem cells; (c) retention of the DNA parental template strand during divisions in some tissue systems, so that mutations are passed to the daughter differentiating cells and not retained in the parental cell; and (d) stem cell competition, whereby undamaged stem cells outcompete damaged stem cells for residence in the niche. DNA repair mainly occurs within a few days of irradiation, while stem cell competition requires weeks or many months depending on the tissue type. The aforementioned processes may contribute to the differences in carcinogenic radiation risk values between tissues, and may help to explain why a rapidly replicating tissue such as small intestine is less prone to such risk. The processes also provide a mechanistic insight relevant to the LNT model, and the relative and absolute risk models. The radiobiological knowledge also provides a scientific insight into discussions of the dose and dose-rate effectiveness factor currently used in radiological protection guidelines. In addition, the biological information contributes potential reasons for the age-dependent sensitivity to radiation carcinogenesis, including the effects of in-utero exposure.

Epigenetic memory gained by priming with osteogenic induction medium improves osteogenesis and other properties of mesenchymal stem cells.

Mesenchymal stem cells (MSCs) are highly plastic cells that are able to transdifferentiate or dedifferentiate under appropriate conditions. In the present study, we reported here that after in vitro induction of osteogenic differentiation, MSCs could be reverted to a primitive stem cell population (dedifferentiated osteogenic MSCs, De-Os-MSCs) with improved cell survival, colony formation, osteogenic potential, migratory capacity and increased expression of Nanog, Oct4 and Sox2. Most importantly, our results showed great superiority of the De-Os-MSCs over untreated MSCs in ectopic bone formation in vivo. Furthermore, Nanog-knockdown in MSCs could reverse these enhanced properties in De-Os-MSCs in vitro, indicating a central role of Nanog in the transcriptional network. In addition, epigenetic regulations including DNA methylation and histone modifications may play important roles in regulating the de-osteogenic differentiation process. And we found decreased methylation and promoter accrual of activating histone marks, such as H3K4me3 and H4ac on both Nanog and Oct4 gene promoters. Taken together, our study demonstrated that epigenetic memory in De-Os-MSCs gained by priming with osteogenic induction medium favored their differentiation along osteoblastic lineage with improved cell survival and migratory abilities, which may have application potential in enhancing their regenerative capacity in mammals.

Sca-1+ cells from fetal heart with high aldehyde dehydrogenase activity exhibit enhanced gene expression for self-renewal, proliferation, and survival.

Stem/progenitor cells from multiple tissues have been isolated based on enhanced activity of cytosolic aldehyde dehydrogenase (ALDH) enzyme. ALDH activity has emerged as a reliable marker for stem/progenitor cells, such that ALDHbright/high cells from multiple tissues have been shown to possess enhanced stemness properties (self-renewal and multipotency). So far though, not much is known about ALDH activity in specific fetal organs. In this study, we sought to analyze the presence and activity of the ALDH enzyme in the stem cell antigen-1-positive (Sca-1+) cells of fetal human heart. Biochemical assays showed that a subpopulation of Sca-1+ cells (15%) possess significantly high ALDH1 activity. This subpopulation showed increased expression of self-renewal markers compared to the ALDHlow fraction. The ALDHhigh fraction also exhibited significant increase in proliferation and pro-survival gene expression. In addition, only the ALDHhigh and not the ALDHlow fraction could give rise to all the cell types of the original population, demonstrating multipotency. ALDHhigh cells showed increased resistance against aldehyde challenge compared to ALDHlow cells. These results indicate that ALDHhigh subpopulation of the cultured human fetal cells has enhanced self-renewal, multipotency, high proliferation, and survival, indicating that this might represent a primitive stem cell population within the fetal human heart.

Dopaminergic-Like Neurons Derived from Oral Mucosa Stem Cells by Developmental Cues Improve Symptoms in the Hemi-Parkinsonian Rat Model

Achieving safe and readily accessible sources for cell replacement therapy in Parkinson’s disease (PD) is still a challenging unresolved issue. Recently, a primitive neural crest stem cell population (hOMSC) was isolated from the adult human oral mucosa and characterized in vitro and in vivo. In this study we assessed hOMSC ability to differentiate into dopamine-secreting cells with a neuronal-dopaminergic phenotype in vitro in response to dopaminergic developmental cues and tested their therapeutic potential in the hemi-Parkinsonian rat model. We found that hOMSC express constitutively a repertoire of neuronal and dopaminergic markers and pivotal transcription factors. Soluble developmental factors induced a reproducible neuronal-like morphology in the majority of hOMSC, downregulated stem cells markers, upregulated the expression of the neuronal and dopaminergic markers that resulted in dopamine release capabilities. Transplantation of these dopaminergic-induced hOMSC into the striatum of hemi-Parkinsonian rats improved their behavioral deficits as determined by amphetamine-induced rotational behavior, motor asymmetry and motor coordination tests. Human TH expressing cells and increased levels of dopamine in the transplanted hemispheres were observed 10 weeks after transplantation. These results demonstrate for the first time that soluble factors involved in the development of DA neurons, induced a DA phenotype in hOMSC in vitro that significantly improved the motor function of hemiparkinsonian rats. Based on their neural-related origin, their niche accessibility by minimal-invasive procedures and their propensity for DA differentiation, hOMSC emerge as an attractive tool for autologous cell replacement therapy in PD.

Correlated miR-mRNA expression signatures of mouse hematopoietic stem and progenitor cell subsets predict "Stemness" and "Myeloid" interaction networks.

Several individual miRNAs (miRs) have been implicated as potent regulators of important processes during normal and malignant hematopoiesis. In addition, many miRs have been shown to fine-tune intricate molecular networks, in concert with other regulatory elements. In order to study hematopoietic networks as a whole, we first created a map of global miR expression during early murine hematopoiesis. Next, we determined the copy number per cell for each miR in each of the examined stem and progenitor cell types. As data is emerging indicating that miRs function robustly mainly when they are expressed above a certain threshold (∼100 copies per cell), our database provides a resource for determining which miRs are expressed at a potentially functional level in each cell type. Finally, we combine our miR expression map with matched mRNA expression data and external prediction algorithms, using a Bayesian modeling approach to create a global landscape of predicted miR-mRNA interactions within each of these hematopoietic stem and progenitor cell subsets. This approach implicates several interaction networks comprising a “stemness” signature in the most primitive hematopoietic stem cell (HSC) populations, as well as “myeloid” patterns associated with two branches of myeloid development.

CD34 stem-cell marker and multidrug resistance proteins P-gp and BCRP in SEGA

The resistance of human malignancy to multiple chemotherapeutic agents remains a major obstacle in cancer therapy due to in part to increased expression of ATP-binding cassette (ABC) transporters gene family, including “multidrug resistance 1”, and “breast cancer resistant protein”. These proteins are differentially expressed during normal hematopoiesis with the highest levels in primitive bone marrow CD34 stem cell population, and similarly, it was also suggested to occur transiently during neurulation. Subependymal Giant Cell Astrocytoma (SEGA) is a periventricular-low-grade tumor usually associated with tuberous sclerosis complex. We previously described that several multidrug-resistance proteins are overexpressed in brain cortical tubers associated with refractory epilepsy; however, they have not been investigated in SEGA. From a previous reported study of 15 brain specimens of SEGA, 6 randomized cases were selected for the immunostaining with specific monoclonal antibodies to multidrug resistance 1, breast cancer resistant protein and CD34 stem-cell marker. Heterogeneous distributions for these markers were detected with differential immunostaining pattern, showing high immunoreactivity in SEGA cells far of vessels, and low or negative expression in SEGA cells near the vessels. This particular expression pattern of both ABCtransporters, and CD34 antigen could identify different stem-cell subset in SEGA.

Periodontal Stem Cells: a Historical Background and Current Perspectives

In this review we discuss the historical perspective of stem cell populations from oral tissues in light of our current understanding of stem cell biology. Stem cells and their niches have been identified in the periodontium starting from the late 1970s. Applying new criteria for the identification and characterization reveals that oral tissues comprise a multipotent primitive neural crest-like stem cell population capable of differentiating into neural crest derived cell lineages of the cranial-facial zone. This population supplies cells to a more restricted stem cell type with tissue specific epigenetic memory that differentiates into cell lineages characteristic of their tissue origin. We believe that the microenvironment plays an essential role in maintaining stem cell populations and directing their migration and differentiation, and that this factor needs to be considered for utilization of stem cell-based therapy for periodontal regeneration and regenerative dental medicine.

UM171 Is a Novel and Potent Agonist Of Human Hematopoietic Stem Cell Renewal

▪Various combinations of soluble factors have been reported to promote the ex vivo proliferation of human HSCs but few of them are capable of promoting self-renewal. Characterization of StemRegenin (SR1), an aryl hydrocarbon receptor (AhR) antagonist that promotes expansion of CD34+ cells ex vivo, provided a proof of principle that low molecular weight (LMW) compounds have the ability to modulate cellular pathways possibly leading to HSC expansion. In an attempt to identify novel putative agonists of HSC self-renewal, we tested a library comprising > 5,000 LMW molecules in a phenotypical screen based on in vitro expansion of CD34+CD45RA- cells. Eight hits were identified, the majority of which were AhR antagonists. Validation studies identified one hit synthesized in our institute which potently expanded human CD34+CD45RA- cord blood cells ex vivo. Importantly this molecule does not antagonize AhR. Structure activity relationship (SAR) generated a compound called UM171 which is 14-25 folds more potent than the starting molecule with an EC50 of 8-15 nM on CD34+ human cord blood (CB). Using the optimal dose of this analog, CD34+, CD34+CD45RA-, and CD34+CD38-CD90+CD45RA-CD49f+ were expanded 115±15, 62±3 and 121±6 fold respectively over the input values after 12 day culture in fed-batch system. The expansion of these phenotypic HSC populations was enhanced, in an additive manner by including SR1. Washout of UM171 led to a rapid loss of the CD34+CD45RA- population suggesting that its effect is reversible. UM171 is not a mitogen and cannot compensate for the absence of cytokines added to the media (Flt3L, SCF, and TPO). Our cell division studies revealed no effect of the compound on division rate compared to control indicating that UM171 cultured cells retain the CD34+CD45RA- phenotype. Apoptosis and cell phonotype analysis showed significantly less cell differentiation and cell death for UM171 cultured cells compared to controls or SR1 supplemented cultures suggesting that the compound acts by anti-differentiation/apoptosis. Similar to SR1, UM171 treated cells showed a 75±20 fold net increase in the numbers of multilineage colony forming cell (CFU-GEMM) and 150±20 fold when both compounds were present in the 12 day culture.To further understand the impact of UM171 on more primitive cell compartments, we transplanted 100, 500, 1000 and 10,000 uncultured CB CD34+ cells and their progeny after a 12-day culture (with or without UM171) in NSG mice and verified the human hematopoietic reconstitution in their bone marrow 20 weeks later. Mice transplanted with UM171 expanded cells showed a much higher level of human CD45 engraftment compared to uncultured or control expanded cells. The NSG Repopulating Cells (NRC) showed a net expansion ex vivo only in the presence of UM171 with values ranging from 3.1 to 5.5 fold increase over input value in 6 independent experiments. Interestingly, the co-treatment with SR1 did not significantly enhance the impact of UM171 on NRC expansion. In these fed-batch conditions SR1 treatment maintained the NRC population at input values. Importantly, we observed a linear relationship between the number of UM171-treated cells transplanted in vivo and level of human CD45 engraftment. Moreover, at limit dilution, these cells were capable of human myeloid and lymphoid differentiation in NSG BM environment suggesting that UM171 expanded cells remain multipotent. Together, our studies have identified a novel compound which truly expands human progenitors while amplifying the more primitive stem cell population. UM171 has successfully passed toxicity studies and is in the final phase of GMP production for a phase I study which will be launched shortly. In addition, preliminary evidence suggests that UM171 strikingly enhances gene transfer efficiency to primitive human CD34 CB cells. Disclosures:No relevant conflicts of interest to declare.

Stem cells of the lamina propria of human oral mucosa and gingiva develop into mineralized tissues in vivo

To characterize the mineralized tissue formed constitutively in the supracalvarial region of scid mice by a primitive stem cell population (hOMSC) derived from the lamina propria of the human oral mucosa and gingiva. Fibrin-hOMSC constructs were cultured for 14 days at which time point they were analysed for the expression of osteoblastic/cementoblastic markers and implanted between the skin and calvaria bones into scid mice. After 8 weeks, the animals were sacrificed and the implantation sites analysed. Two-week-old cultures of fibrin-hOMSC constructs expressed osteogenic/cementogenic markers at the gene level. Macroscopic and radiographic examinations revealed mineralized masses at the implantation sites of fibrin-hOMSC constructs. Histology, histochemistry and immunofluorescence showed mineralized masses consisting of avascular cellular and acellular matrices that stained positively for collagen, Ca, cementum attachment protein, cementum protein 1, bone sialoprotein, alkaline phosphatase, osteocalcin, amelogenin and ameloblastin. Positive anti-human nuclear antigen indicated the human origin of the cells. Atomic force microscopy depicted long prismatic structures organized in lamellar aggregates. Within the limitation of this study, the results indicate for the first time that fibrin-hOMSC constructs are endowed with the constitutive capacity to develop into mineralized tissues that exhibit certain similarities to cementum and bone.

Cystic Hepatic Lesions in c-Kit Negative Systemic Mastocytosis

Purpose: Systemic mastocytosis (SM) is a rare disorder characterized by infiltration of mast cells in visceral organs. We present a 70 year old male with a c-kit negative mutation and aggressive SM presenting as multiple cystic liver lesions and non-cirrhotic portal hypertension. He had presented with worsening dyspnea on exertion, increased abdominal girth and weight loss. He had no history of alcohol, tobacco, IV drug use, transfusion, tattoos or familial liver disease. On exam, he had a fluid wave and hepatomegaly. Labs showed a WBC 3.9 × 10(9)/L with 35% monocytes, hemoglobin 9.7 g/dL, platelets 110 × 10(9)/L, alkaline phosphatase 201 U/L and INR of 1.3. Electrophoresis revealed a polyclonal gammopathy. Viral and autoimmune etiologies were excluded. Ascitic fluid showed a SAAG of 1.6 with total protein of 5 g/L and 89 neutrophils. Cytology showed histiocytes and mesothelial cells. Serum tryptase level was 150 ng/dL (nl<11.5). Imaging showed splenomegaly, ascites, recanalization of the umbilical vein, innumerable cystic liver lesions without arterial enhancement and multiple lytic rib lesions. Bone marrow biopsy revealed hypercellular marrow with an infiltrate of atypical spindle cells, compromising 30-40% of the cellularity. Immunohistochemical staining of the atypical cells was positive for CD117, mast cell tryptase, and CD25. Liver biopsy showed a similar infiltrate of atypical spindle cells, involving the portal areas and dispersed through the sinusoids with associated focal fibrosis. He was treated with one cycle of cladribine with persistent pancytopenia and no response in his bone and hepatic involvement. Genomic analysis, following the initiation of chemotherapy, revealed the absence of the D816V KIT mutation. The patient was enrolled in a trial with midostaurin (PKC412). Discussion: In a majority of adult cases of mastocytosis, including cases with hepatic manifestations, the somatic mutation Asp816Val (D816V) has been described in the catalytic domain of the KIT proto-oncogene. As in our case, patients with negative KIT mutations do not respond to imatinib mesylate. In the progression of mastocytosis, the KIT mutant D816V may disappear as a result of the clonal selection and transformation of more malignant subclones derived from a primitive neoplastic stem cell population. In our patient, the loss of the D816V mutation is possibly explained by clonal selection subsequent to the chemotherapeutic induction. The radiologic findings of cystic liver lesions are uncommon but may be related to obstructed sinusoids from infiltrated mast cells leading to portal hypertension.