Tayloring cell populations for neurodegenerative diseases

Neurological disorders are increasing in prevalence world- wide, and interest in stem cell therapies for these amictions has increased over the past two decades. While many neu- rological injuries are too devastating for the repair capabil- ities of endogenous neural stem cells （NSCs） an alternative is to harvest stem cells from a donor and grow them in vitro, to be used later as a donor source for transplantation. Many research groups have already done this, first using animal models and now using clinical trial participants. Despite the regular flow of publications about cell replace- ment therapies for central nervous system （CNS） disorders, there is still a scarcity of clinically-relevant reports of effi- cacy. The capability of donor cells to undergo ample site-di- rected differentiation and functional integration seems to be lacking （Andressen, 2013）. So, while stem cells do have properties that are suited for repair of the injured CNS, a primary remaining question is how these cells can best be grafted to produce long-term functional benefit to the host environment. Moreover, among the challenges in neural cell transplantation is controlling the ultimate characteris- tics of grafted cells, pertaining to their survival, phenotypes and performance.

FSPAM:A Feature Construction Method to Identifying Cell Populations in ScRNA-seq Data

The emergence of single-cell RNA-sequencing(scRNA-seq)technology has introduced new information about the structure of cells,diseases,and their associated biological factors.One of the main uses of scRNA-seq is identifying cell populations,which sometimes leads to the detection of rare cell populations.However,the new method is still in its infancy and with its advantages comes computational challenges that are just beginning to address.An important tool in the analysis is dimensionality reduction,which transforms high dimensional data into a meaningful reduced subspace.The technique allows noise removal,visualization and compression of high-dimensional data.This paper presents a new dimensionality reduction approach where,during an unsupervised multistage process,a feature set including high valuable markers is created which can facilitate the isolation of cell populations.Our proposed method,called fusion of the Spearman and Pearson affinity matrices(FSPAM),is based on a graph-based Gaussian kernel.Use of the graph theory can be effective to overcome the challenge of the nonlinear relations between cellular markers in scRNA-seq data.Furthermore,with a proper fusion of the Pearson and Spearman correlation coefficient criteria,it extracts a set of the most important features in a new space.In fact,the FSPAM aggregates the various aspects of cell-to-cell similarity derived from the Pearson and Spearman metrics,and reveals new aspects of cell-to-cell similarity,which can be used to extract new features.The results of the identification of cell populations via k-means++clustering method based on the features extracted from the FSPAM and different datasets of scRNA-seq suggested that the proposed method,regardless of the characteristics that govern each dataset,enjoys greater accuracy and better quality compared to previous methods.

Characterization of Side Cell Populations Obtained from Human Amnion Mesenchymal Cells

Human amnion mesenchymal cells （AMCs） contain muhipotent cells. To enrich such muhipotent stem cells, we applied to AMCs the new method for the isolation of side population （SP） cells used for the enrichment of muhipotent stem cells from many tissues. We succeeded in obtaining SP cells from AMCs （AMC-SP cells）. AMC-SP cells were found in 0.2 % of AMCs, irrespective of the length of pregnant period, ranging from 37 to 40 weeks. Cell cycle analyses suggested that AMC-SP cells belonged to a cell population that proliferated very slowly and/or was in a quiescent state in the amniotic membrane. Upon culturing, they proliferated with 40 to 80 cell doublings. However, they did not form colonies in a soft agarose culture, whereas HepG2 cells, representative human hepatoma cells formed many large colonies. These results suggest that AMC-SP cells that have considerable value for the use of regenerative medicine can be managed safely in vitro.

Single-cell transcriptome reveals core cell populations and androgen-RXFP2 axis involved in deer antler full regeneration

Deer antlers constitute a unique mammalian model for the study of both organ formation in postnatal life and annual full regeneration.Previous studies revealed that these events are achieved through the proliferation and differentiation of antlerogenic periosteum(AP)cells and pedicle periosteum(PP)cells,respectively.As the cells resident in the AP and the PP possess stem cell attributes,both antler generation and regeneration are stem cell-based processes.However,the cell composition of each tissue type and molecular events underlying antler development remain poorly characterized.Here,we took the approach of single-cell RNA sequencing(scRNA-Seq)and identified eight cell types(mainly THY1^(+)cells,progenitor cells,and osteochondroblasts)and three core subclusters of the THY1^(+)cells(SC2,SC3,and SC4).Endothelial and mural cells each are heterogeneous at transcriptional level.It was the proliferation of progenitor,mural,and endothelial cells in the activated antler-lineage-specific tissues that drove the rapid formation of the antler.We detected the differences in the initial differentiation process between antler generation and regeneration using pseudotime trajectory analysis.These may be due to the difference in the degree of stemness of the AP-THY1+and PP-THY1^(+)cells.We further found that androgen-RXFP2 axis may be involved in triggering initial antler full regeneration.Fully deciphering the cell composition for these antler tissue types will open up new avenues for elucidating the mechanism underlying antler full renewal in specific and regenerative medicine in general.

Persistence of side population cells with high drug efflux capacity in pancreatic cancer

AIM: To investigate the persistence of side population (SP) cells in pancreatic cancer and their role and mechanism in the drug resistance. METHODS: The presentation of side population cells in pancreatic cancer cell line PANC-1 and its proportion change when cultured with Gemcitabine, was detected by Hoechst 33342 staining and FACS analysis. The expression of ABCB1 and ABCG2 was detected by real- time PCR in either SP cells or non-SP cells. RESULTS: SP cells do exist in PANC-1, with a median of 3.3% and a range of 2.1-8.7%. After cultured with Gemcitabine for 3 d, the proportion of SP cells increased significantly (3.8% ± 1.9%, 10.7% ± 3.7%, t = 4.616, P = 0.001 < 0.05). ABCB1 and ABCG2 expressed at higher concentrations in SP as compared with non-SP cells (ABCB1: 1.15 ± 0.72, 5.82 ± 1.16, t = 10.839, P = 0.000 < 0.05; ABCG2: 1.16 ± 0.75, 5.48 ± 0.94, t = 11.305, P = 0.000 < 0.05), which may contribute to the efflux of fluorescent staining and drug resistance. CONCLUSION: SP cells with inherently high resistance to chemotherapeutic agents do exist in pancreatic cancers, which may be candidate cancer stem cells contributing to the relapse of the tumor.

A mathematical model of cortical bone remodeling at cellular level under mechanical stimulus

A bone cell population dynamics model for cor- tical bone remodeling under mechanical stimulus is devel- oped in this paper. The external experiments extracted from the literature which have not been used in the creation of the model are used to test the validity of the model. Not only can the model compare reasonably well with these ex- perimental results such as the increase percentage of final values of bone mineral content （BMC） and bone fracture en- ergy （BFE） among different loading schemes （which proves the validity of the model）, but also predict the realtime devel- opment pattern of BMC and BFE, as well as the dynamics of osteoblasts （OBA）, osteoclasts （OCA）, nitric oxide （NO） and prostaglandin E2 （PGE2） for each loading scheme, which can hardly be monitored through experiment. In conclusion, the model is the first of its kind that is able to provide an in- sight into the quantitative mechanism of bone remodeling at cellular level by which bone cells are activated by mechan- ical stimulus in order to start resorption/formation of bone mass. More importantly, this model has laid a solid foun- dation based on which future work such as systemic control theory analysis of bone remodeling under mechanical stimu- lus can be investigated. The to-be identified control mecha- nism will help to develop effective drugs and combined non- pharmacological therapies to combat bone loss pathologies. Also this deeper understanding of how mechanical forces quantitatively interact with skeletal tissue is essential for the generation of bone tissue for tissue replacement purposes in tissue engineering.

Side Population Cells in Human Gallbladder Cancer Cell Line GBC-SD Regulated by TGF-β-induced Epithelial-mesenchymal Transition

Mounting evidence has shown that side population （SP） cells are enriched for cancer stem cells （CSCs） responsible for cancer malignancy. In this study, SP technology was used to isolate a small subpopulation of SP cells in human gallbladder cancer cell line GBC-SD, and SP cells which had superior potential for proliferation in vitro and tumorigenesis in vivo were identified. Importantly, the abundance of GBC-SD SP cells was increased by a transforming growth factor-β （TGF-β）-induced epithelial-mesenchymal transition （EMT）, and this effect was accompanied with a strong up-regulation of ABCG2 mRNA expression, and a decreased sensitivity to mitoxantrone. SP cells were restored upon the removal of TGF-β and the reversion of the cells to an epithelial phenotype, and smad3-specific siRNA reduced SP abundance in response to TGF-β. In conclusion, TGF-β-induced EMT by smad-dependent signaling pathway promotes cancer development and anti-cancer drug resistant phenotype by augmenting the abundance of GBC-SD SP cells, and a better understanding of mechanisms involved in TGF-β-induced EMT may provide a novel strategy for preventing cancer progression.

Isolation and Identification of Cancer Stem-like Cells from Side Population of Human Prostate Cancer Cells

It has been widely verified by various sorting methods that cancer stem cells (CSCs) exist in different types of tumor cells or tissues.However,due to lack of specific stem cell surface markers,CSCs are very difficult to be separated from some cancer cells,which becomes the key barrier of functional studies of CSCs.The sorting method by side population cells (SP) lays a solid foundation for in-depth and comprehensive study of CSCs.To identify the existence of SP in prostate cancer cell lines,we applied flow cytometry sorting by SP to cultures of prostate cancer cell lines (TSU,LnCap,and PC-3),and the cancer stem-like characteristics of SP were verified through experiments in vitro and in vivo.The proportion of SP in TSU cells was calculated to be 1.60%±0.40% (±s),and that in PC-3 and LnCap cells was calculated to be 0.80%±0.05% and 0.60%±0.20%,respectively.The colony formation assay demonstrated that the colony formation rate of SP to non-SP sorted from TSU via flow cytometry was 0.495±0.038 to 0.177±0.029 in 500 cells,0.505±0.026 to 0.169±0.024 in 250 cells,and 0.088±0.016 to 0.043±0.012 in 125 cells respectively.In the in vivo experiments,tumors were observed in all the mice on the 10th day after injecting 50 000 cells subcutaneously in SP group,whereas when 5×106 cells were injected in non-SP group,tumors were developed in only 4 out of 8 mice until the 3rd week before the end of the experiment.Our results revealed that prostate cancer cells contain a small subset of cells,called SP,possessing much greater capacity of colony formation and tumorigenic potential than non-SP.These suggest that SP in prostate cancer cells may play a key role in the self-renewal and proliferation,and have the characteristics of cancer stem-like cells.Dissecting these features will provide a new understanding of the function of prostate CSCs in tumorigenicity and transformation.

Isolation of Side Population Cells and Detection of ABCG2 from SW480

Objective：Side population cells （SP cells） are a new type of stem cells. They mainly express ABCG2/BCRP1 and have the ability to eliminate DNA dye Hoechst33342. Many studies showed that side population cells were able of self-renewal, differentiation and carcinogenesis in cancers. Our investigation aimed at isolation of side population cells and ABCG2 positive subpopulation.from colon cancer cell line SW480 and identification of their characteristics of cancer stem cells. Methods： side population cells and non-side population cells of colon cancer cell line SW480 were isolated with DNA dye Hoechst33342 and their cell cycles were measured by flow cytometry. Expression of ABCG2 of SW480 was measured by immunohistochemistry and immunofluorescence, and its proportion was measured by flow cytometry. Results： SW480 contained 2.29% side population ceils. The fraction of side population ceils decreased greatly to 0.40% by treatment with verapamil. The fraction of side population cells in S-G2M cell cycle was 16.14%, which was much lower than the fraction （34.05%） of non-side population cells in S-G2M. In SW480, ABCG2 positive cells, which proportion was 9.66%, were small, circular or oval, lack of psuedopods, similar to poor differentiation. On the contrary, the ABCG2 negative cells were large, polygonal, with many psuedopods, similar to high differentiation. ConclusJon： our assay identified that side population cells did exist in SW480 and had a quiescence characteristic of stem cells. ABCG2 positive subpopulation occupied about 9.66% of SW480 and may have the ability to promote cell self-renewal and inhibit cell differentiation. Therefore, to isolate ABCG2 positive subpopulation from side population cells may be an alternative to study colorectal cancer stem cells.

Isolation of side population cells from gallbladder carcinoma of human being and the expression of ABCG2 gene

Objective: To investigate whether the side population cells (SP cells) exist in human gallbladder carcinoma cell line and the differences of drug resistance gene ABCG2 expression in SP cells, non-SP cells and GBC-SD cell lines. Methods: Fluorescence activated cell sorter (FACS) was used to sort the SP and non-SP cells from GBC-SD cell line of gallbladder carcinoma of human being. Then, the sorting cells were cultured and detected the expression of ABCG2 gene among the SP cells, non-SP cells and GBC-SD cell lines by using reverse transcription polymerase chain reaction (RT-PCR), immunofluo-rescence chemistry, western blot and flow cytometry techniques. Results: A very small fraction cells (0.64 ± 0.08%) were iso-lated through FACS analysis which had the potency of stem cells and highly expressed ABCG2 gene (89.56 ± 3.86%). On the contrary, there were nearly no expression in non-SP cells (1.32 ± 0.49%) and lower expression in GBC-SD cell line (12.37 ± 1.61%). Conclusion: The side population cells that had the potency of stem cells existed in human gallbladder carcinoma cell line and over-expressed the drug resistance gene ABCG2. They may be play an important role in drug resistance of tumor.

Targeting BCRP/ABCG2 by RNA Interference Enhances the Chemotherapy Sensitivity of Human Colon Cancer Side Population Cells

Relapse and metastasis are frequent in colon cancer and may be linked to stem cell characteristics.This study isolated side population（SP） cells from a colon cancer cell line（Colo-320） and examined their self-renewal and differentiation abilities.Compared to non-SP（NSP） cells,SP colon cancer cells were more tumorigenic in vivo and exhibited more invasive characteristics and a greater ability to form colonies.Additionally,more cells were in G0/G1 phase and more highly expressed the multidrug resistance protein BCRP/ABCG2.We achieved enhanced chemotherapy sensitivity by transfecting SP cells with a hairpin-like,small interfering RNA（si RNA） eukaryotic expression plasmid targeting BCRP/ABCG2.

Side population cells isolated from KATO Ⅲ human gastric cancer cell line have cancer stem cell-like characteristics

AIM: To investigate whether the side population (SP) cells possess cancer stem cell-like characteristics in vitro and the role of SP cells in tumorigenic process in gastric cancer. METHODS: We analyzed the presence of SP cells indifferent human gastric carcinoma cell lines, and then isolated and identified the SP cells from the KATO Ⅲ human gastric cancer cell line by flow cytometry. The clonogenic ability and self-renewal were evaluated by clone and sphere formation assays. The related genes were determined by reverse transcription polymerase chain reaction. To compare tumorigenic ability, SP and non-side population (NSP) cells from the KATO Ⅲ human gastric cancer cell line were subcutaneously injected into nude mice. RESULTS: SP cells from the total population accounted for 0.57% in KATO Ⅲ, 1.04% in Hs-746T, and 0.02% in AGS (CRL-1739). SP cells could grow clonally and have self-renewal capability in conditioned media. The expression of ABCG2, MDRI, Bmi-1 and Oct-4 was different between SP and NSP cells. However, there was no apparent difference between SP and NSP cells when they were injected into nude mice. CONCLUSION: SP cells have some cancer stem celllike characteristics in vitro and can be used for studying the tumorigenic process in gastric cancer.

Fractional Order Nonlinear Bone Remodeling Dynamics Using the Supervised Neural Network

This study aims to solve the nonlinear fractional-order mathematical model(FOMM)by using the normal and dysregulated bone remodeling of themyeloma bone disease(MBD).For themore precise performance of the model,fractional-order derivatives have been used to solve the disease model numerically.The FOMM is preliminarily designed to focus on the critical interactions between bone resorption or osteoclasts(OC)and bone formation or osteoblasts(OB).The connections of OC and OB are represented by a nonlinear differential system based on the cellular components,which depict stable fluctuation in the usual bone case and unstable fluctuation through the MBD.Untreated myeloma causes by increasing the OC and reducing the osteoblasts,resulting in net bone waste the tumor growth.The solutions of the FOMM will be provided by using the stochastic framework based on the Levenberg-Marquardt backpropagation(LVMBP)neural networks(NN),i.e.,LVMBPNN.The mathematical performances of three variations of the fractional-order derivative based on the nonlinear disease model using the LVMPNN.The static structural performances are 82%for investigation and 9%for both learning and certification.The performances of the LVMBPNN are authenticated by using the results of the Adams-Bashforth-Moulton mechanism.To accomplish the capability,steadiness,accuracy,and ability of the LVMBPNN,the performances of the error histograms(EHs),mean square error(MSE),recurrence,and state transitions(STs)will be provided.

The exploration of cell population data in clinical use: Beyond infectious diseases

Cell population data(CPD)is regarded as the fingerprint of a blood cell at a given moment.CPD parameters harbor information associated with cell morphology and can be automatically generated using modern hematological analyzers.Various studies have revealed many unique clinical applications for CPD,especially for infectious diseases,such as sepsis.For example,one monocyte-related CPD parameter is the monocyte distribution width(MDW),which can be generated using a Beckman Coulter hematological analyzer.MDW has received FDA and CE approval for aiding in sepsis diagnosis in adult patients in the emergency department.Additionally,MDW can serve as a diagnostic biomarker in patients infected with SARS-CoV-2.CPD has also been widely explored for possible clinical applications beyond infectious dis-eases,such as for predicting myelodysplastic syndromes,screening for he-matological malignancies,and detecting sterile inflammation.CPD parameter measurements are easily obtained and quite cost-effective,making them practical for clinical use.However,there are some potential drawbacks of CPD parameters.Some pre-analytical conditions can affect CPD values.Further-more,CPD are specific to certain hematological analyzers and the result cannot be transferred between different analyzers.The practical usefulness of CPD reference intervals is also still questionable.In this review,wesummarize the current studies related to CPD and its clinical applications.Additional well-designed clinical studies related to CPD are still expected.

Correlation Analysis of Molecularly-Defined Cortical Interneuron Populations with Morpho-Electric Properties in Layer V of Mouse Neocortex

Cortical interneurons can be categorized into distinct populations based on multiple modalities,including molecular signatures and morpho-electrical(M/E)properties.Recently,many transcriptomic signatures based on single-cell RNA-seq have been identified in cortical interneurons.However,whether different interneuron populations defined by transcriptomic signature expressions correspond to distinct M/E subtypes is still unknown.Here,we applied the Patch-PCR approach to simultaneously obtain the M/E properties and messenger RNA(mRNA)expression of>600 interneurons in layer V of the mouse somatosensory cortex(S1).Subsequently,we identified 11 M/E subtypes,9 neurochemical cell populations(NCs),and 20 transcriptomic cell populations(TCs)in this cortical lamina.Further analysis revealed that cells in many NCs and TCs comprised several M/E types and were difficult to clearly distinguish morpho-electrically.A similar analysis of layer V interneurons of mouse primary visual cortex(V1)and motor cortex(M1)gave results largely comparable to S1.Comparison between S1,V1,and M1 suggested that,compared to V1,S1 interneurons were morpho-electrically more similar to M1.Our study reveals the presence of substantial M/E variations in cortical interneuron populations defined by molecular expression.

Characterization of cancer stem-like cells in the side population cells of human gastric cancer cell line MKN-45

Objective:Side population(SP) cells may play a crucial role in tumorigenesis and the recurrence of cancer.Many kinds of cell lines and tissues have demonstrated the presence of SP cells,including several gastric cancer cell lines.This study is aimed to identify the cancer stem-like cells in the SP of gastric cancer cell line MKN-45.Methods:We used fluorescence activated cell sorting(FACS) to sort SP cells in the human gastric carcinoma cell line MKN-45(cells labeled with Hoechst 33342) and then characterized the cancer stem-like properties of SP cells.Results:This study found that the SP cells had higher clone formation efficiency than major population(MP) cells.Five stemness-related gene expression profiles,including OCT-4,SOX-2,NANOG,CD44,and adenosine triphosphate(ATP)-binding cassette transporters gene ABCG2,were tested in SP and MP cells using quantitative real-time reverse transcription polymerase chain reaction(RT-PCR).Western blot was used to show the difference of protein expression between SP and MP cells.Both results show that there was significantly higher protein expression in SP cells than in MP cells.When inoculated into non-obese diabetic/severe combined immunodeficiency(NOD/SCID) mice,SP cells show higher tumorigenesis tendency than MP cells.Conclusions:These results indicate that SP cells possess cancer stem cell properties and prove that SP cells from MKN-45 are gastric cancer stem-like cells.

Preliminary analysis of stem cell-like cells in human neuroblastoma

Background:Neuroblastoma is an embryonic neoplasm originating from the neural crest with cellular heterogeneity as one of its oncobiological characteristics.This study was undertaken to determine whether human neuroblastoma contains stem ell-like cells.Methods:Twenty patients with neuroblastoma who have been treated in our hospital since January 2005 were divided into pre-operative chemotherapy(10 patients)and non-chemotherapy(10)groups.Tumor specimens of the patients were taken and paraffin sections were made.The expressions of stem cell markers CD133,ABCG2,CD117 and nestin were immunohistochemically detected in the specimens.Neuroblastoma cells were stained with Hoechst 33342 and PI.The side population(SP)cells were analyzed by the fluorescence activated cell sorter.The disparity drug resistance to cisplatin(DDP)of SP and non-SP cells was measured with MTT colorimetric assay.The oncogenicity of SP and non SP cells was identified in nude mice.Results:There was no significant difference in the expression intensity of CD117 and nestin between the two groups of specimens(P>0.05).There was a significant difference between the two groups in terms of the expression intensity of CD133 and ABCG2(P<0.05).The SP cells accounted for 0.2%-1.3%of the total human neuroblastoma cells and were decreased to 0.1%-0.5%after verapamil treatment.The SP and non-SP cells showed disparity in cell growth experiment and drug resistance to DDP.Oncogenicity experiment revealed that nude mice could erupt tumor by an injection of 1×10^(6)SH-SY5Y and WIV SP cells.However,the nude mice could not form tumor by an injection of 1×10^(6)non-SP cells.Conclusion:Neuroblastoma might contain cancer stem cell-like cells.

Clinical significance of CD34^(+)CD117^(dim)/CD34^(+)CD117^(bri) myeloblast-associated gene expression in t(8;21)acute myeloid leukemia

OriginalTranslation t(8;21)(q22;q22)acute myeloid leukemia(AML)is a highly heterogeneous hematological malignancy with a high relapse rate in China.Two leukemic myeloblast populations(CD34^(+)CD117^(dim) and CD34^(+)CD117^(bri))were previously identified in t(8;21)AML,and CD34^(+)CD117^(dim) cell proportion was determined as an independent factor for this disease outcome.Here,we examined the impact of CD34^(+)CD117^(dim)/CD34^(+)CD117^(bri) myeloblast-associated gene expression on t(8;21)AML clinical prognosis.In this study,85 patients with t(8;21)AML were enrolled.The mRNA expression levels of CD34^(+)CD117^(dim)-associated genes(LGALS1,EMP3,and CRIP1)and CD34^(+)CD117^(bri)-associated genes(TRH,PLAC8,and IGLL1)were measured using quantitative reverse transcription PCR.Associations between gene expression and clinical outcomes were determined using Cox regression models.Results showed that patients with high LGALS1,EMP3,or CRIP1 expression had significantly inferior overall survival(OS),whereas those with high TRH or PLAC8 expression showed relatively favorable prognosis.Univariate analysis revealed that CD19,CD34^(+)CD117^(dim) proportion,KIT mutation,minimal residual disease(MRD),and expression levels of LGALS1,EMP3,CRIP1,TRH and PLAC8 were associated with OS.Multivariate analysis indicated that KIT mutation,MRD and CRIP1 and TRH expression levels were independent prognostic variables for OS.Identifying the clinical relevance of CD34^(+)CD117^(dim)/CD34^(+)CD117^(bri) myeloblast-associated gene expression may provide new clinically prognostic markers for t(8;21)AML.