Single Cell Origin of Multilineage Colonies in Culture: Evidence That Differentiation of Multipotent Progenitors and Restriction of Proliferative Potential of Monopotent Progenitors Are Stochastic Processes

In this paper, we report analysis of differentiation in human hemopoietic colonies derived from a single cell. Cord blood mononulear cells and panned My-10 antigen-positive bone marrow and cord blood cells were plated in methylcellulose medium containing erythropoietin and conditioned medium. Initially, we performed mapping studies to identify candidate colony-forming cells. Subsequently, using a micromanipulator, we transferred single cells individually to 35-mm dishes for analysis of colony formation. Cellular composition of the colony was determined by identifying all of the cells in the May-Grunwald-Giemsa stained preparation. Of 150 single candidate cells replated, 63 produced colonies. The incidences of single lineage colonies included 19 erythroid, 17 monocyte-macrophage, and 9 eosinophil colonies. There were 18 mixed hemopoietic colonies consisting of cells in two, three, four, and five lineages in varying combinations. In some instances, we noted the predominance of one lineage and the presence of very small populations of cells in a second or third lineage. These results provide evidence for the single-cell origin of human multilineage hemopoietic colonies, and are consistent with the stochastic model of stem cell differentiation in man. They also indicate that restriction of the proliferative potential of committed progenitors is a stochastic process

Improved methods for the generation of human gene knockout and knockin cell lines

Recent studies have demonstrated the utility of recombinant adeno-associated viral (rAAV) vectors in the generation of human knockout cell lines. The efficiency with which such cell lines can be generated using rAAV, in comparison with more extensively described plasmid-based approaches, has not been directly tested. In this report, we demonstrate that targeting constructs delivered by rAAV vectors were nearly 25-fold more efficient than transfected plasmids that target the same exon. In addition, we describe a novel vector configuration which we term the synthetic exon promoter trap (SEPT). This targeting element further improved the efficiency of knockout generation and uniquely facilitated the generation of knockin alterations. An rAAV-based SEPT targeting construct was used to transfer a mutant CTNNB1 allele, encoding an oncogenic form of β-catenin, from one cell line to another. This versatile method was thus shown to facilitate the efficient integration of small, defined sequence alterations into the chromosomes of cultured human cells

Hematopoietic differentiation of human embryonic stem cells progresses through sequential hematoendothelial, primitive, and definitive stages resembling human yolk sac development

We elucidate the cellular and molecular kinetics of the stepwise differentiation of human embryonic stem cells (hESCs) to primitive and definitive erythromyelopoiesis from human embryoid bodies (hEBs) in serum-free clonogenic assays. Hematopoiesis initiates from CD45 hEB cells with emergence of semiadherent mesodermal-hematoendothelial (MHE) colonies that can generate endothelium and form organized, yolk sac–like structures that secondarily generate multipotent primitive hematopoietic stem progenitor cells (HSPCs), erythroblasts, and CD13+CD45+ macrophages. A first wave of hematopoiesis follows MHE colony emergence and is predominated by primitive erythropoiesis characterized by a brilliant red hemoglobinization, CD71/CD325a (glycophorin A) expression, and exclusively embryonic/fetal hemoglobin expression. A second wave of definitive-type erythroid burst-forming units (BFU-e's), erythroid colony-forming units (CFU-e's), granulocyte-macrophage colony-forming cells (GM-CFCs), and multilineage CFCs follows next from hEB progenitors. These stages of hematopoiesis proceed spontaneously from hEB-derived cells without requirement for supplemental growth factors during hEB differentiation. Gene expression analysis of differentiating hEBs revealed that initiation of hematopoiesis correlated with increased levels of SCL/TAL1, GATA1, GATA2, CD34, CD31, and the homeobox gene-regulating factor CDX4 These data indicate that hematopoietic differentiation of hESCs models the earliest events of embryonic and definitive hematopoiesis in a manner resembling human yolk sac development, thus providing a valuable tool for dissecting the earliest events in human HSPC genesis