Abnormal B-cell development in TIMP-deficient bone marrow.

Bone marrow (BM) is the primary site of hematopoiesis and is responsible for a lifelong supply of all blood cell lineages. The process of hematopoiesis follows key intrinsic programs that also integrate instructive signals from the BM niche. First identified as an erythropoietin-potentiating factor, the tissue inhibitor of metalloproteinase (TIMP) protein family has expanded to 4 members and has widely come to be viewed as a classical regulator of tissue homeostasis. By virtue of metalloprotease inhibition, TIMPs not only regulate extracellular matrix turnover but also control growth factor bioavailability. The 4 mammalian TIMPs possess overlapping enzyme-inhibition profiles and have never been studied for their cumulative role in hematopoiesis. Here, we show that TIMPs are critical for postnatal B lymphopoiesis in the BM. TIMP-deficient mice have defective B-cell development arising at the pro-B-cell stage. Expression analysis of TIMPless hematopoietic cell subsets pointed to an altered B-cell program in the Lineage-Sca-1+c-Kit+ (LSK) cell fraction. Serial and competitive BM transplants identified a defect in TIMP-deficient hematopoietic stem and progenitor cells for B lymphopoiesis. In parallel, reverse BM transplants uncovered the extrinsic role of stromal TIMPs in pro- and pre-B-cell development. TIMP deficiency disrupted CXCL12 localization to LepR+ cells, and increased soluble CXCL12 within the BM niche. It also compromised the number and morphology of LepR+ cells. These data provide new evidence that TIMPs control the cellular and biochemical makeup of the BM niche and influence the LSK transcriptional program required for optimal B lymphopoiesis.

Mammary epithelial cells have lineage-rooted metabolic identities.

Cancer metabolism adapts the metabolic network of its tissue of origin. However, breast cancer is not a disease of a single origin. Multiple epithelial populations serve as the culprit cell of origin for specific breast cancer subtypes, yet our knowledge of the metabolic network of normal mammary epithelial cells is limited. Using a multi-omic approach, here we identify the diverse metabolic programmes operating in normal mammary populations. The proteomes of basal, luminal progenitor and mature luminal cell populations revealed enrichment of glycolysis in basal cells and of oxidative phosphorylation in luminal progenitors. Single-cell transcriptomes corroborated lineage-specific metabolic identities and additional intra-lineage heterogeneity. Mitochondrial form and function differed across lineages, with clonogenicity correlating with mitochondrial activity. Targeting oxidative phosphorylation and glycolysis with inhibitors exposed lineage-rooted metabolic vulnerabilities of mammary progenitors. Bioinformatics indicated breast cancer subtypes retain metabolic features of their putative cell of origin. Thus, lineage-rooted metabolic identities of normal mammary cells may underlie breast cancer metabolic heterogeneity and targeting these vulnerabilities could advance breast cancer therapy.

Rudhira/BCAS3 is essential for mouse development and cardiovascular patterning.

Rudhira/Breast Carcinoma Amplified Sequence 3 (BCAS3) is a cytoskeletal protein that promotes directional cell migration and angiogenesis in vitro and is implicated in human carcinomas and coronary artery disease. To study the role of Rudhira during development in vivo, we generated the first knockout mouse for rudhira and show that Rudhira is essential for mouse development. Rudhira null embryos die at embryonic day (E) 9.5 accompanied by severe vascular patterning defects in embryonic and extra-embryonic tissues. To identify the molecular processes downstream of rudhira, we analyzed the transcriptome of intact knockout yolk sacs. Genome-wide transcriptome analysis showed that Rudhira functions in angiogenesis and its related processes such as cell adhesion, extracellular matrix organization, peptidase activity and TGFß signaling. Since Rudhira is also expressed in endothelial cells (ECs), we further generated Tie2Cre-mediated endothelial knockout (CKO) of rudhira. CKO embryos survive to E11.5 and similar to the global knockout, display gross vascular patterning defects, showing that endothelial Rudhira is vital for development. Further, Rudhira knockdown ECs in culture fail to sprout in a spheroid-sprouting assay, strongly supporting its role in vascular patterning. Our study identifies an essential role for Rudhira in blood vessel remodeling and provides a mouse model for cardiovascular development.

Generation of a constitutively expressing Tetracycline repressor (TetR) human embryonic stem cell line BJNhem20-TetR.

Human embryonic stem cell line BJNhem20-TetR was generated using non-viral method. The construct pCAG-TetRnls was transfected using microporation procedure. BJNhem20-TetR can subsequently be transfected with any vector harbouring a TetO (Tet operator) sequence to generate doxycycline based inducible line. For example, in human embryonic stem cells, the pSuperior based TetO system has been transfected into a TetR containing line to generate OCT4 knockdown cell line (Zafarana et al., 2009). Thus BJNhem20-TetR can be used as a tool to perturb gene expression in human embryonic stem cells.

Generation of OCIAD1 inducible overexpression human embryonic stem cell line: BJNhem20-OCIAD1-Tet-On.

Human embryonic stem cell line BJNhem20-OCIAD1-Tet-On was generated using non-viral method. The constructs pCAG-Tet-On and pTRE-Tight vector driving OCIAD1 expression were transfected using microporation procedure. pCAG-Tet-On cells can be used for inducible expression of any coding sequence cloned into pTRE-Tight vector. For example, in human embryonic stem cells, Tet-On system has been used to generate SOX2 overexpression cell line (Adachi et al., 2010).

Generation of human embryonic stem cell line expressing green fluorescent protein.

Human embryonic stem cell line BJNhem20 pCAG-EGFP was generated using non-viral method (Fig. 1). The construct pCAG-EGFP was transfected using microporation procedure.

Derivation of human embryonic stem cell lines from poor quality embryos.

A serious shortcoming in the derivation of human embryonic stem cell (hESC) lines has been the availability of human embryos. About 60% of human embryos generated by in vitro fertilization (IVF) fail to develop normally and are unusable for fertility treatment. Such embryos often retain sufficient pluripotent cells that can generate genetically normal, pluripotent hESC lines with stable phenotype. We describe here a simple protocol for isolating pluripotent stem cells from abnormally developed grade III human embryos that are an unutilized byproduct of in vitro fertility treatment. Embryos that progress to the blastocyst stage are subjected to immunosurgery or mechanical surgery to isolate the inner cell mass (ICM). Isolated cells are plated on to fibroblast feeders in hESC derivation media. Pluripotent cells that grow from the ICM are isolated mechanically and cultured to obtain a stable hESC line. In this way, we derived two sibling hESC lines BJNhem19 and BJNhem20 that represent the Indian ethnic background and show stable phenotype upon long-term continuous culture of over 225 passages.