In vivo editing of lung stem cells for durable gene correction in mice.

In vivo genome correction holds promise for generating durable disease cures; yet, effective stem cell editing remains challenging. In this work, we demonstrate that optimized lung-targeting lipid nanoparticles (LNPs) enable high levels of genome editing in stem cells, yielding durable responses. Intravenously administered gene-editing LNPs in activatable tdTomato mice achieved >70% lung stem cell editing, sustaining tdTomato expression in >80% of lung epithelial cells for 660 days. Addressing cystic fibrosis (CF), NG-ABE8e messenger RNA (mRNA)-sgR553X LNPs mediated >95% cystic fibrosis transmembrane conductance regulator (CFTR) DNA correction, restored CFTR function in primary patient-derived bronchial epithelial cells equivalent to Trikafta for F508del, corrected intestinal organoids and corrected R553X nonsense mutations in 50% of lung stem cells in CF mice. These findings introduce LNP-enabled tissue stem cell editing for disease-modifying genome correction.

Preparation of selective organ-targeting (SORT) lipid nanoparticles (LNPs) using multiple technical methods for tissue-specific mRNA delivery.

A new methodology termed selective organ targeting (SORT) was recently developed that enables controllable delivery of nucleic acids to target tissues. SORT lipid nanoparticles (LNPs) involve the inclusion of SORT molecules that accurately tune delivery to the liver, lungs and spleen of mice after intravenous administration. Nanoparticles can be engineered to target specific cells and organs in the body by passive, active and endogenous targeting mechanisms that require distinct design criteria. SORT LNPs are modular and can be prepared using scalable, synthetic chemistry and established engineering formulation methods. This protocol provides detailed procedures, including the synthesis of a representative ionizable cationic lipid, preparation of multiple classes of SORT LNPs by pipette, vortex and microfluidic mixing methods, physical characterization, and in vitro/in vivo mRNA delivery evaluation. Depending on the scale of the experiments, the synthesis of the ionizable lipid requires 4-6 d; LNPs can be formulated within several hours; LNP characterization can be completed in 2-4 h; and in vitro/in vivo evaluation studies require 1-14 d, depending on the design and application. Our strategy offers a versatile and practical method for rationally designing nanoparticles that accurately target specific organs. The SORT LNPs generated as described in this protocol can therefore be applied to multiple classes of LNP systems for therapeutic nucleic acid delivery and facilitate the development of protein replacement and genetic medicines in target tissues. This protocol does not require specific expertise, is modular to various lipids within defined physicochemical classes, and should be accomplishable by researchers from various backgrounds.

Long Non-Coding RNA H19 Acts as an Estrogen Receptor Modulator that is Required for Endocrine Therapy Resistance in ER+ Breast Cancer Cells.

BACKGROUND/AIMS: Blocking estrogen signaling with endocrine therapies (Tamoxifen or Fulverstrant) is an effective treatment for Estrogen Receptor-α positive (ER+) breast cancer tumours. Unfortunately, development of endocrine therapy resistance (ETR) is a frequent event resulting in disease relapse and decreased overall patient survival. The long noncoding RNA, H19, was previously shown to play a significant role in estrogen-induced proliferation of both normal and malignant ER+ breast epithelial cells. We hypothesized that H19 expression is also important for the proliferation and survival of ETR cells. METHODS: Here we utilized established ETR cell models; the Tamoxifen (Tam)-resistant LCC2 and the Fulvestrant and Tam cross-resistant LCC9 cells. Gain and loss of H19 function were achieved through lentiviral transduction as well as pharmacological inhibitors of the Notch and c-Met receptor signaling pathways. The effects of altered H19 expression on cell viability and ETR were assessed using three-dimensional (3D) organoid cultures and 2D co-cultures with low passage tumour-associated fbroblasts (TAFs). RESULTS: Here we report that treating ETR cells with Tam or Fulvestrant increases H19 expression and that it's decreased expression overcomes resistance to Tam and Fulvestrant in these cells. Interestingly, H19 expression is regulated by Notch and HGF signaling in the ETR cells and pharmacological inhibitors of Notch and c-MET signaling together significantly reverse resistance to Tam and Fulvestrant in an H19-dependent manner in these cells. Lastly, we demonstrate that H19 regulates ERα expression at the transcript and protein levels in the ETR cells and that H19 protects ERα against Fulvestrant-mediated downregulation of ERα protein. We also observed that blocking Notch and the c-MET receptor signaling also overcomes Fulvestrant and Tam resistance in 3D organoid cultures by decreasing ERα and H19 expression in the ETR cells. CONCLUSION: In endocrine therapy resistant breast cancer cells Fulvestrant is ineffective in decreasing ERα levels. Our data suggest that in the ETR cells, H19 expression acts as an ER modulator and that its levels and subsequently ERα levels can be substantially decreased by blocking Notch and c-MET receptor signaling. Consequently, treating ETR cells with these pharmacological inhibitors helps overcome resistance to Fulvestrant and Tamoxifen.

A robust cell culture system for large scale feeder cell-free expansion of human breast epithelial progenitors.

BACKGROUND: Normal human breast epithelial cells are maintained by the proliferation and differentiation of different human breast epithelial progenitors (HBEPs). However, these progenitor subsets can only be obtained at low frequencies, limiting their further characterization. Recently, it was reported that HBEPs can be minimally expanded in Matrigel cocultures with stromal feeder cells. However, variability of generating healthy feeder cells significantly impacts the effective expansion of HBEPs. METHODS: Here, we report a robust feeder cell-free culture system for large-scale expansion of HBEPs in two-dimensional cultures. RESULTS: Using this cell culture system HBEPs can be exponentially expanded as bulk cultures. Moreover, purified HBEP subtypes can also be separately expanded using our cell culture system. The expanded HBEPs retain their undifferentiated phenotype and form distinct epithelial colonies in colony forming cell assays. CONCLUSIONS: The availability of a culture system enabling the large-scale expansion of HBEPs facilitates their application to screening platforms and other cell-based assays.

In vivo evidence supporting a metastasis suppressor role for Stard13 (Dlc2) in ErbB2 (Neu) oncogene induced mouse mammary tumors.

Overexpression of dominant oncogenes and the loss of tumor suppressor genes are basic genetic events in the acquisition of the malignant phenotype. The erb-b2 receptor tyrosine kinase 2 (ERBB-2) proto-oncogene is overexpressed in 20-30% of human breast cancers. The StAR related lipid transfer domain containing 13 gene (STARD13), also known as Deleted in Liver Cancer-2 (DLC-2), maps to chromosome band 13q12.3 and is frequently downregulated in human cancers, including 72% of breast cancers. It encodes a RhoGAP protein with sterile α motif (SAM) and StAR-related lipid transfer (START) domains. The objective of this study was to determine if loss of Stard13 plays a role in mammary tumor progression using transgenic mice expressing the activated ErbB-2 (Neu) oncogene and Cre recombinase (NIC) in mammary epithelium under transcriptional control of the murine mammary tumor virus (MMTV) promoter (MMTV-NIC). These mice were crossed with a conditional Stard13 knockout mouse (floxed exon 3), resulting in simultaneous Neu expression and Stard13 deletion, specifically in the mammary epithelium. We found that loss of Stard13 did not alter tumor growth nor significantly modify overall survival and tumor free survival. However, there was an increase in the total number of lung metastases in the Stard13 heterozygous or homozygous mice compared with the parental MMTV-NIC strain. Altogether our results indicate that Stard13 acts as a metastasis suppressor rather than a tumor suppressor gene, in Neu oncogene induced mammary tumorigenesis.

Breast Cancers Activate Stromal Fibroblast-Induced Suppression of Progenitors in Adjacent Normal Tissue.

Human breast cancer cells are known to activate adjacent "normal-like" cells to enhance their own growth, but the cellular and molecular mechanisms involved are poorly understood. We now show by both phenotypic and functional measurements that normal human mammary progenitor cells are significantly under-represented in the mammary epithelium of patients' tumor-adjacent tissue (TAT). Interestingly, fibroblasts isolated from TAT samples showed a reduced ability to support normal EGF-stimulated mammary progenitor cell proliferation in vitro via their increased secretion of transforming growth factor ß. In contrast, TAT fibroblasts promoted the proliferation of human breast cancer cells when these were co-transplanted in immunodeficient mice. The discovery of a common stromal cell-mediated mechanism that has opposing growth-suppressive and promoting effects on normal and malignant human breast cells and also extends well beyond currently examined surgical margins has important implications for disease recurrence and its prevention.

The Deleted in Liver Cancer 1 (Dlc1) tumor suppressor is haploinsufficient for mammary gland development and epithelial cell polarity.

BACKGROUND: Deleted in Liver Cancer 1 (Dlc1) is a tumor suppressor gene, which maps to human chromosome 8p21-22 and is found frequently deleted in many cancers including breast cancer. The promoter of the remaining allele is often found methylated. The Dlc1 gene encodes a RhoGAP protein that regulates cell proliferation, migration and inhibits cell growth and invasion when restored in Dlc1 deficient tumor cell lines. This study focuses on determining the role of Dlc1 in normal mammary gland development and epithelial cell polarity in a Dlc1 gene trapped (gt) mouse. METHODS: Mammary gland whole mount preparations from 10-week virgin heterozygous Dlc1(gt/+) gene-trapped mice were compared with age-matched wild type (WT) controls. Hematoxylin-Eosin (H&E) and Masson's Trichrome staining of histological sections were carried out. Mammary glands from Dlc1(gt/+) mice and WT controls were enzymatically digested with collagenase and dispase and then cultured overnight to deplete hematopoietic and endothelial cells. The single cell suspensions were then cultured in Matrigel for 12 days. To knockdown Dlc1 expression, primary WT mammary epithelial cells were infected with short hairpin (sh) RNA expressing lentivirus or with a scrambled shRNA control. RESULTS: Dlc1(gt/+) mice showed anomalies in the mammary gland that included increased ductal branching and deformities in terminal end buds and branch points. Compared to the WT controls, Masson's Trichrome staining showed a thickened stromal layer with increased collagen deposition in mammary glands from Dlc1(gt/+) mice. Dlc1(gt/+) primary mammary epithelial cells formed increased solid acinar spheres in contrast with WT and scrambled shRNA control cells, which mostly formed hollow acinar structures when plated in 3D Matrigel cultures. These solid acinar structures were similar to the acinar structures formed when Dlc1 gene expression was knocked down in WT mammary cells by shRNA lentiviral transduction. The solid acinar structures were not due to a defect in apoptosis as determined by a lack of detectible cleaved caspase 3 antibody staining. Primary mammary cells from Dlc1(gt/+) mice showed increased RhoA activity compared with WT cells. CONCLUSIONS: The results illustrate that decreased Dlc1 expression can disrupt the normal cell polarization and mammary ductal branching. Altogether this study suggests that Dlc1 plays a role in maintaining normal mammary epithelial cell polarity and that Dlc1 is haploinsufficient.

Estrogen regulates luminal progenitor cell differentiation through H19 gene expression.

Although the role of estrogen signaling in breast cancer development has been extensively studied, the mechanisms that regulate the indispensable role of estrogen in normal mammary gland development have not been well studied. Because of the unavailability of culture system to maintain estrogen-receptor-positive (ERα(+)) cells in vitro, the molecular mechanisms that regulate estrogen/ERα signaling in the normal human breast are unknown. In the present study, we examined the effects of estrogen signaling on ERα(+) human luminal progenitors using a modified matrigel assay and found that estrogen signaling increased the expansion potential of these progenitors. Furthermore, we found that blocking ERα attenuated luminal progenitor expansion and decreased the luminal colony-forming potential of these progenitors. Additionally, blocking ERα decreased H19 expression in the luminal progenitors and led to the development of smaller luminal colonies. We further showed that knocking down the H19 gene in the luminal progenitors significantly decreased the colony-forming potential of the luminal progenitors, and this phenotype could not be rescued by the addition of estrogen. Lastly, we explored the clinical relevance of the estrogen-H19 signaling axis in breast tumors and found that ERα(+) tumors exhibited a higher expression of H19 as compared with ERα(-) tumors and that H19 expression showed a positive correlation with ERα expression in those tumors. Taken together, the present results indicate that the estrogen-ERα-H19 signaling axis plays a role in regulating the proliferation and differentiation potentials of the normal luminal progenitors and that this signaling network may also be important in the development of ER(+) breast cancer tumors.

Pesticide induced alterations in marrow physiology and depletion of stem and stromal progenitor population: an experimental model to study the toxic effects of pesticide.

Long-term exposure of agriculturally used organochloride and organophosphate pesticides have been shown to cause long-lasting hematotoxicity and increased incidence of aplastic anemia in humans. The mechanisms involved in pesticide induced hematotoxicity and the features of toxicity that may play a major role in bone marrow suppression are not known. The aim of the present study was to investigate the hematological consequences of pesticide exposure in swiss albino mice exposed to aqueous mixture of common agriculturally used pesticides for 6 h/day, 5 days/week for 13 weeks. After the end of last exposure, without a recovery period, the strong hematotoxic effect of pesticide was assessed in mice with long-term bone marrow explant culture (LTBMC-Ex) system and cell colony forming assays. Bone marrow explant culture from the pesticide exposed group of mice failed to generate a supportive stromal matrix and did not produce adequate number of hematopoietic cells and found to contain largely the adipogenic precursors. The decreased cell colony numbers in the pesticide exposed group indicated defective maturational and functional status of different marrow cell lineages. As a whole, exposure of mice to the mixture of pesticides reduced the total number of bone marrow cells (granulocytes are the major targets of pesticide toxicity), hematopoietic, and non-hematopoietic progenitor cells and most of the hematological parameters. Replication of primitive stem/progenitor cells in the marrow was decreased following pesticide exposure with G0/G1-phase arrest of most of the cells. The progenitor cells showed decreased percentage of cells in S/G2/M-phase. The increased apoptosis profile of the marrow progenitors (Increased CD95 expression) and primitive stem cells (High Annexin-V positivity on Sca1+ cells) with an elevated intracellular cleaved caspase-3 level on the Sca1+ bone marrow cells provided the base necessary for explaining the deranged bone marrow microenvironmental structure which was evident from scanning electron micrographs. These results clearly indicate a strong, long lasting toxic effect of pesticides on the bone marrow microenvironment and different microenvironmental components which ultimately leads to the formation of a degenerative disease like aplastic anemia.

Pesticide induced marrow toxicity and effects on marrow cell population and on hematopoietic stroma.

Long term inhalation of toxic pesticides used for the domestic and industrial purposes have been shown to cause moderate to severe hematotoxicity and increased incidence of several marrow degenerative diseases, specifically hypoplastic bone marrow failure condition in humans. The progression of pesticide induced hematotoxicity and the exact underlying mechanisms of toxicity that play major role in limiting normal hematopoiesis are not quite well explained. In this present study, we have developed an animal model of hypoplastic bone marrow failure following pesticide exposure to show the deleterious effects of toxic pesticides on mouse hematopoietic system. Here we have presented the results of studying long-term marrow explant culture, IL-2, IL-3 and IL-5 receptors expression profile, fibroblast colony forming unit (CFU-F), hematopoietic progenitor cell colony formation and caspase-3 expression by the bone marrow cells. We have also identified the expression levels of several extracellular apoptosis markers (CD95/Fas) and intracellular apoptosis inducer proteins (pASK1, pJNK, caspase-3 and cleaved caspase-3) in the bone marrow cells of pesticide exposed mice. The long-term marrow explant culture demonstrated the impairment in proliferation of the stromal cells/stromal fibroblasts in culture. Decreased IL-2, IL-3 and IL-5 receptors expression profile essentially hinted at the suppressed cytokine activity in the pesticide exposed marrow. CFU-F analysis showed the defect in functional maturation of the stromal fibroblasts. The decreased hematopoietic progenitor cell colony formation indicated the toxicity induced inhibition of cellular proliferation and functional maturation of hematopoietic stem/progenitor cells in pesticide exposed marrow. We have detected a sharp increase in the expression levels of both the extracellular Fas-antigen and intracellular apoptosis inducer proteins in the bone marrow cells of pesticide exposed mice that explained well, the apoptosis pathway involved following marrow toxicity. The decreased proliferation and functional maturation of marrow stromal cells and hematopoietic progenitors with subsequent increase in marrow cellular apoptosis following pesticide toxicity provided the base necessary for explaining the increased incidence of hypoplastic bone marrow failure in humans exposed to moderate to high concentrations of pesticides.

The biology of human breast epithelial progenitors.

Current evidence suggests that similar to other tissues in the human body mammary epithelia cells are being maintained by the unique properties of stem cells, undifferentiated as well as lineage-restricted progenitors. Because of their longevity, proliferation and differentiation potentials these primitive breast epithelial cells are likely targets of transforming mutations that can cause them to act as cancer initiating cells. In this context, understanding the molecular mechanisms that regulate the normal functions of the human breast epithelial stem cells and progenitors and how alterations to these same mechanisms can confer a cancer stem cell phenotype on these rare cell populations is crucial to the development of new and more effective therapies again breast cancer. This review article will examine the current state of knowledge about the isolation and characterization of human breast epithelial progenitors and their relevance to breast cancer research.

Leukemic stromal hematopoietic microenvironment negatively regulates the normal hematopoiesis in mouse model of leukemia.

BACKGROUND AND OBJECTIVE: Leukemic microenvironment has a major role in the progression of leukemia. Leukemic cells can induce reversible changes in microenvironmental components, especially the stromal function which results in improved growth conditions for maintaining the malignant leukemic cells. This study aimed to investigate the survival advantage of leukemic cells over normal hematopoietic cells in stromal microenvironment in long term. METHODS: The mice were injected intraperitoneally with N-N' ethylnitrosourea (ENU) to induce leukemia; the mice received injection of normal saline were used as control. At 180 days after ENU induction, the mice were killed and the bone marrows were cultured for 19 days. Colony-forming assays were used to analyze the formation of various cell colonies. The expression of Sca-1, CD146, VEGFR2, CD95, pStat3, pStat5, and Bcl-xL in marrow cells were detected by flow cytometry. RESULTS: Long-term leukemic bone marrow culture showed abnormal elongated stromal fibroblasts with almost absence of normal hematopoietic cells. Adherent cell colonies were increased, but CFU-F and other hematopoietic cell colonies were significantly decreased in leukemia group (P<0.001). Primitive progenitor-specific Sca-1 receptor expression was decreased with subsequent increased expression of CD146 and VEGFR-2 in leukemic bone marrow cells. Decreased Fas antigen expression with increased intracellular pStat3, pStat5 and Bcl-xL proteins were observed in leukemic bone marrow cells. CONCLUSIONS: Stromal microenvironment shows altered morphology and decreased maturation in leukemia. Effective progenitor cells are decreased in leukemia with increased leukemia-specific cell population. Leukemic microenvironment plays a role in promoting and maintaining the leukemic cell proliferation and survivability in long term.

Primitive Sca-1 Positive Bone Marrow HSC in Mouse Model of Aplastic Anemia: A Comparative Study through Flowcytometric Analysis and Scanning Electron Microscopy.

Self-renewing Hematopoietic Stem Cells (HSCs) are responsible for reconstitution of all blood cell lineages. Sca-1 is the "stem cell antigen" marker used to identify the primitive murine HSC population, the expression of which decreases upon differentiation to other mature cell types. Sca-1(+) HSCs maintain the bone marrow stem cell pool throughout the life. Aplastic anemia is a disease considered to involve primary stem cell deficiency and is characterized by severe pancytopenia and a decline in healthy blood cell generation system. Studies conducted in our laboratory revealed that the primitive Sca-1(+) BM-HSCs (bone marrow hematopoietic stem cell) are significantly affected in experimental Aplastic animals pretreated with chemotherapeutic drugs (Busulfan and Cyclophosphamide) and there is increased Caspase-3 activity with consecutive high Annexin-V positivity leading to premature apoptosis in the bone marrow hematopoietic stem cell population in Aplastic condition. The Sca-1(bright), that is, "more primitive" BM-HSC population was more affected than the "less primitive" BM-HSC Sca-1(dim ) population. The decreased cell population and the receptor expression were directly associated with an empty and deranged marrow microenvironment, which is evident from scanning electron microscopy (SEM). The above experimental evidences hint toward the manipulation of receptor expression for the benefit of cytotherapy by primitive stem cell population in Aplastic anemia cases.

Alteration in marrow stromal microenvironment and apoptosis mechanisms involved in aplastic anemia: an animal model to study the possible disease pathology.

Aplastic anemia (AA) is a heterogeneous disorder of bone marrow failure syndrome. Suggested mechanisms include a primary stem cell deficiency or defect, a secondary stem cell defect due to abnormal regulation between cell death and differentiation, or a deficient microenvironment. In this study, we have tried to investigate the alterations in hematopoietic microenvironment and underlying mechanisms involved in such alterations in an animal model of drug induced AA. We presented the results of studying long term marrow culture, marrow ultra-structure, marrow adherent and hematopoietic progenitor cell colony formation, flowcytometric analysis of marrow stem and stromal progenitor populations and apoptosis mechanism involved in aplastic anemia. The AA marrow showed impairment in cellular proliferation and maturation and failed to generate a functional stromal microenvironment even after 19 days of culture. Ultra-structural analysis showed a degenerated and deformed marrow cellular association in AA. Colony forming units (CFUs) were also severely reduced in AA. Significantly decreased marrow stem and stromal progenitor population with subsequently increased expression levels of both the extracellular and intracellular apoptosis inducer markers in the AA marrow cells essentially pointed towards the defective hematopoiesis; moreover, a deficient and apoptotic microenvironment and the microenvironmental components might have played the important role in the possible pathogenesis of AA.

Phenotypic alteration of bone marrow HSC and microenvironmental association in experimentally induced leukemia.

Leukemia is a heterogeneous disorder of bone marrow (BM) failure syndrome where normal hematopoiesis gets altered due to transformation of either the normal hematopoietic cell or the hematopoietic microenvironment or both. Scientists have tried for decades to understand leukemia development in the context of therapeutic strategies. The existence of "leukemic stem cells" and their possible role in leukemogenesis have only recently been identified and it has changed the perspective with regard to new approaches for treating the disease. However the relationship between leukemic stem cells (LSCs) and leukemogenesis requires further investigation. In this present study, we have experimentally induced leukemia in mice by means of N-N' Ethylnitrosourea (ENU) to investigate the alterations in normal bone marrow cellular phenotype and associated changes in the stromal hematopoietic microenvironment under the event of leukemic disease progression. We have identified a significant decrease in the normal HSC phenotype in terms of Sca1 and c-kit receptor expression and subsequent sharp increase in certain leukemic cell specific receptor expression like CD123, CXCR4 and CD44 in the leukemic bone marrow. The decreased HSC receptor (Sca1 and c-kit) expression profile with concurrent increase in the expression of leukemic cell specific receptors (CD123, CXCR4, CD44) by the bone marrow cells of leukemic mice may account for the possible transformation of the normal hematopoietic cells that is necessary for the disease initiation and progression. Some of these receptors like CXCR4 and CD44 are also known to play an important role in maintaining leukemic cells and their complex crosstalk with the surrounding stromal microenvironment. Thus up-regulation in CXCR4 and CD44 receptor expression essentially pointed towards the stroma dependent surveillance of the leukemic bone marrow cells in leukemia. Leukemic bone marrow cells documented a rapid generation of stromal feeder layer in culture. The rapid stroma generation further supported the fact that leukemic stromal microenvironment gets altered in possible ways to support leukemic cell generation and fueling leukemogenesis. The study presented here, has tried to hint at exploring new therapeutic strategies by not only identifying the expression profile of cell surface receptors unique to cells involved in leukemic progression but also targeting the specific components of the stromal microenvironment that would facilitate therapeutic management of the disease.

Effects of inorganic arsenic on bone marrow hematopoietic cells: an emphasis on apoptosis and Sca-1/c-Kit positive population.

Apoptosis, proliferation and differentiation are balanced molecular processes which may alter their pattern during environmental insults. Arsenic is an environmental pollutant, ranks 20(th) in abundance in the earth crust, 14(th) in sea water and 12(th) in the human body. Millions of people worldwide are chronically exposed to arsenic often due to naturally occurring arsenic in ground water. Hematopoietic stem cells within the bone marrow are the source of all haematopoietic cell lineages and are essential for tissue development throughout the life. In this experimental study, we have evaluated the impact of arsenic, on blood and blood forming cells by the changes in their cellular morphology, immune functional capacity, alteration of bone marrow CD34 positive stem/progenitors and changes in the phenotype of Sca-1, c-Kit dual positive primitive stem cell population. The study revealed that arsenic has a significant effect on bone marrow and hematopoietic stem cells, their immune capacity and upregulation of death process, all indicative of impairment in differentiation suggesting presence of deregulation in their precursors by arsenic toxicity.

The bone marrow stem stromal imbalance--a key feature of disease progression in case of myelodysplastic mouse model.

Myelodysplastic syndromes (MDSs) represent a spectrum of disorders that are generally thought to arise from a defective hematopoietic stem cell leading to clonal, dysregulated hematopoiesis. Although it is generally agreed that the marrow microenvironment plays a role in the biology of MDS, it is unclear whether this represents an intrinsically abnormal stromal compartment derived from the MDS clone. Hematopoiesis requires cooperation between progenitors and a variety of functionally and phenotypically different cell types that form the bone marrow stroma. Stromal abnormalities suspected to contribute to the pathology of bone marrow disorder with impaired hematopoiesis. Several studies on human MDS bone marrow microenvironment revealed functional alteration and increased cellular apoptosis thus contribute to the pathology of the disease progression. In this present study, we have investigated alterations in the hematopoietic microenvironment and underlying mechanisms involved in the disease progression of MDS animal model. We presented the results of bone marrow single cell culture study, Long-term bone marrow adherent culture study (LTBMC) and their functional efficacy, flowcytometric characterization of stem (Scal+c-kit+) and stromal (Scal+CD44+) progenitor cell population and expression level of extracellular apoptosis marker (Annexin v) in the bone marrow cells of MDS animal model. Bone marrow single cell culture study of MDS animal showed impairment in the normal cellular generation, proliferation and presence of apoptic cells. Long-term liquid Bone marrow stromal cell colony formation assay from MDS bone marrow cells showed significant difference in the colony formation and their maintenance than the control groups of animals. Immune functional capacity of the bone marrow stromal cells through cell mediated immune (CMI) parameter study denoted defects in the stromal microenvironment. Decreased expression of bone marrow long-term primitive hematopoietic population and stromal progenitor population depicted bone marrow abnormality in case of MDS animal model, which bears significant correlation with high expression level of apoptosis marker in the bone marrow cells. From the above experimental study we tried to highlight the abnormal bone marrow microenvironment and alteration in the bone marrow cell surface marker expression, which could be the probable mechanism of evolution and disease progression in case of MDS animal model.

Sca-1 expression pattern in the mouse limbal epithelium and its association with cell cycle.

Stem Cell Antigen-1 or Sca-1 is a cell surface receptor protein commonly used to detect adult murine haematopoietic stem cell population. Outside the haematopoietic system Sca-1 is similarly expressed in stem and progenitor cells in a wide variety of tissues and organs such as skeletal muscle, mammary gland, prostate, heart, liver and dermis. Thus Sca-1 has become a candidate marker in the search of tissue specific stem cells. The maintenance of a healthy corneal epithelium is achieved by a unique population of stem cell located in the limbal epithelial region. This limbal epithelium mainly contains limbal epithelial stem cells and its immediate progenitor early transient amplifying cells (e-TAC) which have self renewal capacity. As stem cells in other organs have been identified by their expression of Sca-1, in our study we wanted to determine whether this antigen could be present in the limbal epithelial region which contains stem cell population by using immunofluorescence through flow cytometric analysis of Sca-1 and its association with the cell cycle.

An animal model of chronic aplastic bone marrow failure following pesticide exposure in mice.

The wide use of pesticides for agriculture, domestic and industrial purposes and evaluation of their subsequent effect is of major concern for public health. Human exposure to these contaminants especially bone marrow with its rapidly renewing cell population is one of the most sensitive tissues to these toxic agents represents a risk for the immune system leading to the onset of different pathologies. In this experimental protocol we have developed a mouse model of pesticide(s) induced hypoplastic/aplastic marrow failure to study quantitative changes in the bone marrow hematopoietic stem cell (BMHSC) population through flowcytometric analysis, defects in the stromal microenvironment through short term adherent cell colony (STACC) forming assay and immune functional capacity of the bone marrow derived cells through cell mediated immune (CMI) parameter study. A time course dependent analysis for consecutive 90 days were performed to monitor the associated changes in the marrow's physiology after 30(th), 60(th) and 90(th) days of chronic pesticide exposure. The peripheral blood showed maximum lowering of the blood cell count after 90 days which actually reflected the bone marrow scenario. Severe depression of BMHSC population, immune profile of the bone marrow derived cells and reduction of adherent cell colonies pointed towards an essentially empty and hypoplastic marrow condition that resembled the disease aplastic anemia. The changes were accompanied by splenomegaly and splenic erythroid hyperplasia. In conclusion, this animal model allowed us a better understanding of clinico-biological findings of the disease aplastic anemia following toxic exposure to the pesticide(s) used for agricultural and industrial purposes.

Kinetic impairment of haemopoietic stem cells in experimentally induced leukemia and aplastic anemia: an inverse correlation.

The production of blood cells from bone marrow (BM) hematopoietic stem cells (HSC) is regulated by a number of cytokines and growth factors that influence cell survival; differentiation, proliferation and apoptosis in health and supposedly, such mechanisms are deregulated in diseased conditions. As far as cellular kinetics is concerned HSCs are relatively quiescent in adults, have the ability to replicate symmetrically and asymmetrically and predictably exhibit multi-lineage hematopoietic reconstitution potential. HSC drive hematopoiesis and homeostasis by contracting and expanding the pool of hematopoietic cells in the bone marrow. In mouse they can be identified immunophenotypically as Sca1+ c-kit cells. In aplastic anemia a drastic decline in the marrow efficacy to produce mature blood cells leads to bone marrow failure. In contrast, in leukemia hyper stimulated marrow leads to deregulated differentiation of immature hematopoietic stem cells with increased self-proliferation potential. In our experimental set up, we induced aplastic anemia by injecting busulfan and cyclophosphamide and leukemia by N-N' ethylnitrosourea intraperitoneally in inbred swiss albino mice. Indeed, HSCs and haematopoietic progenitor cells (HPCs) are vulnerable target for such disease oriented dysregulation which bears close correlation with the bone marrow microenvironmental damage. The present study aims at evaluating the possible mechanism(s) of deregulation in the bone marrow physiology with special reference to HSC surface receptor expression, cellular granularity, cell cycle status and overall marrow architecture. The investigations made so far revealed an interesting correlation between disease initiation and specific cytokinetic involvement of HSC in the BM microenvironment with particular reference to leukemia and aplastic anemia.