Purification of rare hematopoietic stem cell(s) (HSC) to homogeneity is required to study their self-renewal, differentiation, phenotype, and homing. Long-term repopulation (LTR) of irradiated hosts and serial transplantation to secondary hosts represent the gold standard for demonstrating self-renewal and differentiation, the defining properties of HSC. We show that rare cells that home to bone marrow can LTR primary and secondary recipients. During the homing, CD34 and SCA-1 expression increases uniquely on cells that home to marrow. These adult bone marrow cells have tremendous differentiative capacity as they can also differentiate into epithelial cells of the liver, lung, GI tract, and skin. This finding may contribute to clinical treatment of genetic disease or tissue repair.

Adult cancers may derive from stem or early progenitor cells 1,2 . Epigenetic modulation of gene expression is essential for normal function of these early cells, but is highly abnormal in cancers, which often exhibit aberrant promoter CpG island hypermethylation and transcriptional silencing of tumor suppressor genes and pro-differentiation factors [3][4][5] . We find that, for such genes, both normal and malignant embryonic cells generally lack the gene DNA hypermethylation found in adult cancers. In embryonic stem (ES) cells, these genes are held in a "transcription ready" state mediated by a "bivalent" promoter chromatin pattern consisting of the repressive polycomb group (PcG) H3K27me mark plus the active mark, H3K4me. However, embryonic carcinoma (EC) cells add two key repressive marks, H3K9me2 and H3K9me3, both associated with DNA hypermethylated genes in adult cancers [6][7][8] . We hypothesize that cell chromatin patterns and transient silencing of these important growth regulatory genes in stem or progenitor cells of origin for cancer may leave these genes vulnerable to aberrant DNA hypermethylation and heritable gene silencing in adult tumors.Correspondence may be addressed to S.B.B. at sbaylin@jhmi.edu. Competing Interests Statement. The commercial rights to the MSP technique belong to Oncomethylome. S.B.B and J.G.H. serve as consultants to Oncomethylome and is entitled to royalties from any commercial use of this procedure. Epigenetic gene silencing and associated promoter CpG island DNA hypermethylation are prevalent in all cancer types, and provide an alternative mechanism to mutations by which tumor suppressor genes may be inactivated within a cancer cell [3][4][5] . These epigenetic changes may precede genetic changes in pre-malignant cells and foster the accumulation of additional genetic and epigenetic hits 9 . Adult cancers may derive from stem or early progenitor cells 1, 2 , and epigenetic modulation of gene expression is essential for normal function of these early cells. We now explore whether DNA hypermethylation and heritable silencing of groups of genes in adult tumor initiation and progression might reflect chromatin properties for these genes associated with a stem or precursor cell of origin. NIH Public AccessWe compared the epigenetic status of a group of genes frequently hypermethylated and silenced in adult cancers ( Fig. 1-all (Fig. 1). Among the genes studied, 13 of 29 (45%) are hypermethylated in a single line, HCT-116, of adult colon cancer, but none are hypermethylated in ES cells, and only 3% and 7% were completely methylated in the Tera-1 and Tera-2 EC lines, respectively. Thus, the key epigenetic parameter of promoter CpG island hypermethylation which is common in a large group of genes in adult cancer cells does not seem to be a common feature of EC cells.In murine ES cells, many developmental genes are maintained in a state of low transcriptional activity and are available for transcription increases or decreases when differentiation cues are received 11 . Our s...

A low-oxygenic niche in bone marrow limits reactive oxygen species (ROS) production, thus providing long-term protection for hematopoietic stem cells (HSCs) from ROS stress. Although many approaches have been used to enrich HSCs, none has been designed to isolate primitive HSCs located within the lowoxygenic niche due to difficulties of direct physical access. Here we show that an early HSC population that might reside in the niche can be functionally isolated by taking advantage of the relative intracellular ROS activity. Many attributes of primitive HSCs in the low-oxygenic osteoblastic niche, such as quiescence, and calcium receptor, N-cadherin, Notch1, and p21 are higher in the ROS low population. Intriguingly, the ROS low population has a higher self-renewal potential. In contrast, significant HSC exhaustion in the ROS high population was observed following serial transplantation, and expression of activated p38 mitogen-activated protein kinase (MAPK) and mammalian target of rapamycin (mTOR) was higher in this population. Importantly, treatment with an antioxidant, a p38 inhibitor, or rapamycin was able to restore HSC function in the ROS high population. Thus, more potent HSCs associated with the lowoxygenic niche can be isolated by selecting for the low level of ROS expression. IntroductionStem cells have a unique mechanism to cope with the cumulative reactive oxygen species (ROS) load, which involves increased antioxidant defenses and unique redox-dependent effects on growth and differentiation. [1][2][3][4][5] Hematopoietic stem cells (HSCs) and the supporting cells of the stem-cell niche are predominantly located in a low-oxygen milieu of the bone marrow, which allows long-term protection from ROS-related oxidative stress. 4,6,7 In the osteoblastic niche, the lowest end of an oxygen gradient within the bone marrow, 6,[8][9][10] HSCs remain quiescent and in contact with osteoblasts, 11 whereas in the relatively more oxygenic vascular niche, due to the proximity to blood circulation, stem cells actively proliferate and differentiate, [12][13][14][15] which might increase the intracellular ROS level. 1 In the osteoblastic niche, the calcium-sensing receptor (CaR) plays a critical role in localizing HSCs to the endosteal surface, although it does not affect HSC homing, 16 and osteoblast-derived factors have been suggested to improve survival of umbilical cord-derived HSCs under hypoxic conditions. 10 HSCs have been enriched using a variety of techniques, including cell-surface antigen selection, 17 elutriation, 18 pharmacologic manipulation, 19 and supravital dye, 20 as well as intracellular enzyme content. 21 However, none of these methods was designed to isolate quiescent HSCs located within the osteoblastic niche, which is generally considered to house the most primitive HSCs. 6,[8][9][10][11]14,22,23 Since it is difficult to experimentally access viable cells within the osteoblastic niche, these HSCs have not been directly or indirectly isolated by taking advantage of any of the specific properties of ...

We have tested the hypothesis that multipotential hemopoietic stem and progenitor cells prime several different lineage-affiliated programs of gene activity prior to unilineage commitment and differentiation. Using single cell RT-PCR we show that erythroid (p-globin) and myeloid (myeloperoxidase) gene expression programs can be initiated by the same cell prior to exclusive commitment to the erythroid or granulocytic lineages. Furthermore, the multipotential state is characterized by the coexpression of several lineage-affiliated cytokine receptors. These data support a model of hemopoietic lineage specification in which unilineage commitment is prefaced by a "promiscuous" phase of multilineage locus activation.

No abstract

Both plasticity and cell fusion have been suggested to have a role in germ-layer switching. To understand the mechanisms underlying cell fate changes, we have examined a highly enriched population of hematopoietic stem cells (HSCs) in vitro or in vivo in response to injury for liver-specific phenotypic and functional changes. Here we show that HSCs become liver cells when cocultured with injured liver separated by a barrier. Chromosomal analyses and tissue-specific gene and/or protein expression show that microenvironmental cues rather than fusion are responsible for conversion in vitro. We transplanted HSCs into liver-injured mice and observed that HSCs convert into viable hepatocytes with increasing injury. Notably, liver function was restored 2-7 d after transplantation. We conclude that HSCs contribute to the regeneration of injured liver by converting into functional hepatocytes without fusion.

Human embryonic stem cells (hESCs) self-renew indefinitely while maintaining pluripotency. The molecular mechanism underlying hESCs self-renewal and pluripotency is poorly understood. To identify the signaling pathway molecules that maintain the proliferation of hESCs, we performed a microarray analysis comparing an aneuploid H1 hESC line (named H1T) versus euploid H1 hESC line because the H1T hESC line demonstrates a selfrenewal advantage while maintaining pluripotency. We find differential gene expression for the Nodal/Activin, fibroblast growth factor (FGF), Wnt, and Hedgehog (Hh) signaling pathways in the H1T line, which implicates each of these molecules in maintaining the undifferentiated state, whereas the bone morphogenic protein (BMP) and Notch pathways could promote hESCs differentiation. Experimentally, we find that Activin A is necessary and sufficient for the maintenance of self-renewal and pluripotency of hESCs and supports long-term feeder and serum-free growth of hESCs. We show that Activin A induces the expression of Oct4, Nanog, Nodal, Wnt3, basic FGF, and FGF8 and suppresses the BMP signal. Our data indicates Activin A as a key regulator in maintenance of the stemness in hESCs. This finding will help elucidate the complex signaling network that maintains the hESC phenotype and function.

Human induced pluripotent stem cells (iPSCs) are a potential source of hepatocytes for liver transplantation to treat end-stage liver disease. In vitro differentiation of human iPSCs into hepatic cells has been achieved using a multistage differentiation protocol, but whether these cells are functional and capable of engrafting and regenerating diseased liver tissue is not clear. We show that human iPSC-derived hepatic cells at various differentiation stages can engraft the liver in a mouse transplantation model. Using the same differentiation and transplantation protocols, we also assessed the ability of human iPSCs derived from each of the three developmental germ layer tissues (that is, ectoderm, mesoderm, and endoderm) to regenerate mouse liver. These iPSC lines, with similar but distinct global DNA methylation patterns, differentiated into multistage hepatic cells with an efficiency similar to that of human embryonic stem cells. Human hepatic cells at various differentiation stages derived from iPSC lines of different origins successfully repopulated the liver tissue of mice with liver cirrhosis. They also secreted human-specific liver proteins into mouse blood at concentrations comparable to that of proteins secreted by human primary hepatocytes. Our results demonstrate the engraftment and liver regenerative capabilities of human iPSC-derived multistage hepatic cells in vivo and suggest that human iPSCs of distinct origins and regardless of their parental epigenetic memory can efficiently differentiate along the hepatic lineage.