Our understanding of how mesodermal tissue is formed has been limited by the absence of specific and reliable markers of early mesoderm commitment. We report that mesoderm commitment from human embryonic stem cells (hESCs) is initiated by epithelialto-mesenchymal transition (EMT) as shown by gene expression profiling and by reciprocal changes in expression of the cell surface proteins, EpCAM/CD326 and NCAM/CD56. Molecular and functional assays reveal that the earliest CD326 − CD56 + cells, generated from hESCs in the presence of activin A, BMP4, VEGF, and FGF2, represent a multipotent mesoderm-committed progenitor population. CD326 − CD56 + progenitors are unique in their ability to generate all mesodermal lineages including hematopoietic, endothelial, mesenchymal (bone, cartilage, fat, fibroblast), smooth muscle, and cardiomyocytes, while lacking the pluripotency of hESCs. CD326 − CD56 + cells are the precursors of previously reported, more lineage-restricted mesodermal progenitors. These findings present a unique approach to study how germ layer specification is regulated and offer a promising target for tissue engineering.CD326 | CD56 | epithelial-to-mesenchymal transition | mesenchyme | hematopoiesis

Rodent bone marrow cells can contribute to liver. If these findings are applicable to humans, marrow stem cells could theoretically be harvested from a patient and used to repair his/her damaged liver. To explore this potential, CD34(+) or highly purified CD34(+)CD38(-)CD7(-) human hematopoietic stem cells from umbilical cord blood and bone marrow were transplanted into immunodeficient mice. One month after transplantation, carbon tetrachloride (CCl(4)) was administered into the mice to induce liver damage and hepatocyte proliferation. Mice were analyzed in comparison with CCl(4)-injured mice that did not receive transplants and noninjured controls that received transplants with the same stem cell populations, one month after liver damage. Human-specific albumin mRNA and protein were expressed in the mouse liver and human albumin was detected in the serum of mice that had received CCl(4) injury. Human alpha-fetoprotein was never expressed, but in some mice, human cytokeratin 19 was expressed, which may indicate bile duct development in addition to the albumin-secreting hepatocyte-like cells. Human albumin was not expressed in the starting stem cell populations in injured mice that did not receive transplants nor in noninjured mice that had received transplants of human stem cells. Human albumin expression was detected only in CCl(4)-treated mice that received transplants of human stem cells, and recovery was increased by administration of human hepatocyte growth factor 48 hours after the CCl(4)-mediated liver injury. Our studies provide evidence that human "hematopoietic" stem/progenitor cell populations have the capacity to respond to the injured liver microenvironment by inducing albumin expression.

Key Points Perivascular cells maintain HSPCs ex vivo.

The expression of CD10 has long been used to define human lymphoid commitment. We report a unique lymphoid-primed population in human bone marrow that was generated from hematopoietic stem cells (HSCs) before the onset of CD10 expression and B cell commitment. This subset was identified by high expression of the homing molecule L-selectin (CD62L). CD10−CD62Lhi progenitors possessed full lymphoid and monocytic potential, but lacked erythroid potential. Gene expression profiling placed the CD10−CD62Lhi population at an intermediate stage of differentiation between HSCs and lineage-negative (Lin−) CD34+CD10+ progenitors. L-selectin was expressed on immature thymocytes and its ligands were expressed at the cortico-medullary junction, suggesting a possible role in thymic homing. These studies identify the earliest stage of lymphoid priming in human bone marrow.

ABSTRACT

The standard approach to assess hematopoietic stem cell (HSC) engraftment in experimental bone marrow transplantation models relies on detection of donor hematopoietic cells in host bone marrow following death; this approach provides data from only a single time point after transplantation for each animal. In vivo bioluminescence imaging was therefore explored as a method to gain a dynamic, longitudinal profile of human HSC engraftment in a living xenogeneic model. Luciferase expression using a lentiviral vector allowed detection of distinctly different patterns of engraftment kinetics from human CD34 ؉ and CD34 ؉ CD38 ؊ populations in the marrow NOD/SCID/␤2m null mice. Imaging showed an early peak (day 13) of engraftment from CD34 ؉ cells followed by a rapid decline in signal. Engraftment from the more primitive CD34 ؉ CD38 ؊ population was relatively delayed but by day 36 increased to significantly higher levels than those from CD34 ؉ cells (P < .05). Signal intensity from CD34 ؉ CD38 ؊ -engrafted mice continued to increase during more than 100 days of analysis. Flow cytometry analysis of bone marrow from mice after death demonstrated that levels of 1% donor cell engraftment could be readily detected by bioluminescence imaging; higher engraftment levels corresponded to higher image signal intensity.

The identity and lineage potential of the cells that initiate thymopoiesis remain controversial. The goal of these studies was to determine, at a clonal level, the immunophenotype and differentiation pathways of the earliest progenitors in human thymus. Although the majority of human CD34 ؉ lin ؊ thymocytes express high levels of CD7, closer analysis reveals that a continuum of CD7 expression exists, and 1% to 2% of progenitors are CD7 ؊ . CD34 ؉ lin ؊ thymocytes were fractionated by CD7 expression and tested for lineage potential in B-lymphoid, T-

Mucopolysaccharidosis type I (MPS I) is a lysosomal glycosaminoglycan (GAG) storage disorder caused by deficiency of alpha-l-iduronidase (IDUA). In this study, we evaluated the potential to perform gene therapy for MPS I by direct in vivo injection of a lentiviral vector, using an IDUA gene knockout murine model. We compared the efficacy in newborn versus young adult MPS I mice of a single intravenous injection of the lentiviral vector. The extent of transduction was dose-dependent, with the liver receiving the highest level of vector, but other somatic organs reaching almost the same level. The phenotypic manifestations of disease were partially improved in the mice treated as young adults, but were nearly normalized at every end-point measured in the mice treated as neonates. In the neonatally treated mice, the expressed IDUA activity resulted in decreased GAG storage, prevention of skeletal abnormalities, a more normal gross appearance, and improved survival. Most strikingly, significant levels of IDUA enzyme were produced in the brain of mice treated as neonates, with transduction of neurons at high levels. The sustained expression of enzymatically active IDUA in multiple organs had a significant beneficial effect on the phenotypic abnormalities of MPS I, which may be translated to clinical gene therapy of patients with Hurler disease.