Lymphoid priming in human bone marrow begins before expression of CD10 with upregulation of L-selectin.

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

Pleiotropic Roles of VEGF in the Microenvironment of the Developing Thymus.

Neonatal life marks the apogee of murine thymic growth. Over the first few days after birth, growth slows and the murine thymus switches from fetal to adult morphology and function; little is known about the cues driving this dramatic transition. In this study, we show for the first time (to our knowledge) the critical role of vascular endothelial growth factor (VEGF) on thymic morphogenesis beyond its well-known role in angiogenesis. During a brief window a few days after birth, VEGF inhibition induced rapid and profound remodeling of the endothelial, mesenchymal and epithelial thymic stromal compartments, mimicking changes seen during early adult maturation. Rapid transcriptional changes were seen in each compartment after VEGF inhibition, including genes involved in migration, chemotaxis, and cell adhesion as well as induction of a proinflammatory and proadipogenic signature in endothelium, pericytes, and mesenchyme. Thymocyte numbers fell subsequent to the stromal changes. Expression patterns and functional blockade of the receptors VEGFR2 and NRP1 demonstrated that VEGF mediates its pleiotropic effects through distinct receptors on each microenvironmental compartment of the developing mouse thymus.

Perivascular support of human hematopoietic stem/progenitor cells.

Hematopoietic stem and progenitor cells (HSPCs) emerge and develop adjacent to blood vessel walls in the yolk sac, aorta-gonad-mesonephros region, embryonic liver, and fetal bone marrow. In adult mouse bone marrow, perivascular cells shape a "niche" for HSPCs. Mesenchymal stem/stromal cells (MSCs), which support hematopoiesis in culture, are themselves derived in part from perivascular cells. In order to define their direct role in hematopoiesis, we tested the ability of purified human CD146(+) perivascular cells, as compared with unfractionated MSCs and CD146(-) cells, to sustain human HSPCs in coculture. CD146(+) perivascular cells support the long-term persistence, through cell-to-cell contact and at least partly via Notch activation, of human myelolymphoid HSPCs able to engraft primary and secondary immunodeficient mice. Conversely, unfractionated MSCs and CD146(-) cells induce differentiation and compromise ex vivo maintenance of HSPCs. Moreover, CD146(+) perivascular cells express, natively and in culture, molecular markers of the vascular hematopoietic niche. Unexpectedly, this dramatic, previously undocumented ability to support hematopoietic stem cells is present in CD146(+) perivascular cells extracted from the nonhematopoietic adipose tissue.

Engineering the human thymic microenvironment to support thymopoiesis in vivo.

A system that allows manipulation of the human thymic microenvironment is needed both to elucidate the extrinsic mechanisms that control human thymopoiesis and to develop potential cell therapies for thymic insufficiency. In this report, we developed an implantable thymic microenvironment composed of two human thymic stroma populations critical for thymopoiesis; thymic epithelial cells (TECs) and thymic mesenchyme (TM). TECs and TM from postnatal human thymi were cultured in specific conditions, allowing cell expansion and manipulation of gene expression, before reaggregation into a functional thymic unit. Human CD34+ hematopoietic stem and progenitor cells (HSPC) differentiated into T cells in the aggregates in vitro and in vivo following inguinal implantation of aggregates in immune deficient mice. Cord blood HSPC previously engrafted into murine bone marrow (BM), migrated to implants, and differentiated into human T cells with a broad T cell receptor repertoire. Furthermore, lentiviral-mediated expression of vascular endothelial growth factor in TM enhanced implant size and function and significantly increased thymocyte production. These results demonstrate an in vivo system for the generation of T cells from human HSPC and represent the first model to allow manipulation of gene expression and cell composition in the microenvironment of the human thymus.

Novel pathways to erythropoiesis induced by dimerization of intracellular C-Mpl in human hematopoietic progenitors.

The cytokine thrombopoietin (Tpo) plays a critical role in hematopoiesis by binding to the extracellular domain and inducing homodimerization of the intracellular signaling domain of its receptor, c-Mpl. Mpl homodimerization can also be accomplished by binding of a synthetic ligand to a constitutively expressed fusion protein F36VMpl consisting of a ligand binding domain (F36V) and the intracellular signaling domain of Mpl. Unexpectedly, in contrast to Tpo stimulation, robust erythropoiesis is induced after dimerization of F36VMpl in human CD34+ progenitor cells. The goal of this study was to define the hematopoietic progenitor stages at which dimerization of intracellular Mpl induces erythropoiesis and the downstream molecular events that mediate this unanticipated effect. Dimerization (in the absence of erythropoietin and other cytokines) in human common myeloid progenitors and megakaryocytic erythroid progenitors caused a significant increase in CD34+ cells (p < .01) and induced all stages of erythropoiesis including production of enucleated red blood cells. In contrast, erythropoiesis was not seen with Tpo stimulation. CD34+ cell expansion was the result of increased cell cycling and survival (p < .05). Microarray profiling of CD34+ cells demonstrated that a unique transcriptional pattern is activated in progenitors by F36VMpl dimerization. Ligand-inducible dimerization of intracellular Mpl in human myeloerythroid progenitors induces progenitor expansion and erythropoiesis through molecular mechanisms that are not shared by Tpo stimulation of endogenous Mpl.

Mapping the first stages of mesoderm commitment during differentiation of human embryonic stem cells.

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 epithelial-to-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.

Rapid thymic reconstitution following bone marrow transplantation in neonatal mice is VEGF-dependent.

Age-related differences in thymic function influence the rapidity of T cell reconstitution following hematopoietic stem cell transplantation (HSCT). In adults, thymic reconstitution is delayed until after marrow engraftment is established, and is significantly improved by approaches that increase marrow chimerism, such as pretransplantation irradiation. In contrast, we show that neonatal mice undergo more rapid and efficient thymic reconstitution than adults, even when bone marrow (BM) engraftment is minimal and in the absence of pretransplantation radiation. We have previously shown that the neonatal thymus produces high levels of vascular endothelial growth factor (VEGF) that drives angiogenesis locally. In this report, we show that inhibition of VEGF prior to HSCT prevents rapid thymic reconstitution in neonates, but has no effect on thymic reconstitution in adults. These data suggest that the early radiation-independent thymic reconstitution unique to the neonatal host is mediated through VEGF, and reveals a novel pathway that might be targeted to improve immune reconstitution post-HSCT.

Effects of sublethal irradiation on patterns of engraftment after murine bone marrow transplantation.

Attempts to reduce the toxicity of hematopoietic stem cell transplantation have led to the use of various immunosuppressive, yet nonmyeloablative preparative regimens that often include low-dose irradiation. To determine the effects of low-dose irradiation on the dynamics of donor cell engraftment after bone marrow transplantation (BMT), we coupled standard endpoint flow cytometric analysis with in vivo longitudinal bioluminescence imaging performed throughout the early (<10 days) and late (days 10-90) post-BMT periods. To exclude the contribution of irradiation on reducing immunologic rejection, severely immune-deficient mice were chosen as recipients of allogeneic bone marrow. Flow cytometric analysis showed that sublethal doses of total body irradiation (TBI) significantly increased long-term (14 weeks) donor chimerism in the bone marrow compared with nonirradiated recipients (P < .05). Bioluminescence imaging demonstrated that the effect of TBI (P < .001) on chimerism occurred only after the first 7 days post-BMT. Flow cytometric analysis on day 3 showed no increase in the number of donor cells in irradiated bone marrow, confirming that sublethal irradiation does not enhance marrow chimerism early after transplantation. Local irradiation also significantly increased late (but not early) donor chimerism in the irradiated limb. Intrafemoral injection of donor cells provided efficient early chimerism in the injected limb, but long-term systemic donor chimerism was highest with i.v. administration (P < .05). Overall, the combination of TBI and i.v. administration of donor cells provided the highest levels of long-term donor chimerism in the marrow space. These findings suggest that the major effect of sublethal irradiation is to enhance long-term donor chimerism by inducing proliferative signals after the initial phase of homing.

VEGF-mediated cross-talk within the neonatal murine thymus.

Although the mechanisms of cross-talk that regulate the hematopoietic and epithelial compartments of the thymus are well established, the interactions of these compartments with the thymic endothelium have been largely ignored. Current understanding of the thymic vasculature is based on studies of adult thymus. We show that the neonatal period represents a unique phase of thymic growth and differentiation, marked by endothelium that is organized as primitive, dense networks of capillaries dependent on vascular endothelial growth factor (VEGF). VEGF dependence in neonates is mediated by significantly higher levels of both VEGF production and endothelial VEGF receptor 2 (VEGF-R2) expression than in the adult thymus. VEGF is expressed locally in the neonatal thymus by immature, CD4(-)CD8(-) "double negative" (DN) thymocytes and thymic epithelium. Relative to adult thymus, the neonatal thymus has greater thymocyte proliferation, and a predominance of immature thymocytes and cortical thymic epithelial cells (cTECs). Inhibition of VEGF signaling during the neonatal period results in rapid loss of the dense capillaries in the thymus and a marked reduction in the number of thymocytes. These data demonstrate that, during the early postnatal period, VEGF mediates cross-talk between the thymocyte and endothelial compartments of the thymus.

Regulated expansion of human pancreatic beta-cells.

Although pancreatic beta-cell transplantation may serve as a potential cure for diabetes mellitus (DM), limited donor tissue availability poses a major challenge. Thus, there is a great demand to find new sources for pancreatic beta-cells. Here, we present a lentiviral vector-based approach to achieve beta-cell proliferation through the beta-cell-specific activation of the hepatocyte growth factor (HGF)/cmet signaling pathway. The methodology is based on the beta-cell-specific expression of a ligand-inducible, chimeric receptor (F36Vcmet), under transcriptional control of the promoter from the human insulin gene, and its ability to induce HGF/cmet signaling in the presence of a synthetic ligand (AP20187). High transduction efficiency of human pancreatic islets was achieved utilizing this approach with chimeric receptor expression confined to the beta-cell population. In addition, specific proliferation of human pancreatic beta-cells was induced utilizing this approach. Selective, regulated beta-cell expansion may help to provide greater availability of cells for transplantation in patients with DM.

A CD133-expressing murine liver oval cell population with bilineage potential.

Although oval cells are postulated to be adult liver stem cells, a well-defined phenotype of a bipotent liver stem cell remains elusive. The heterogeneity of cells within the oval cell fraction has hindered lineage potential studies. Our goal was to identify an enriched population of bipotent oval cells using a combination of flow cytometry and single cell gene expression in conjunction with lineage-specific liver injury models. Expression of cell surface markers on nonparenchymal, nonhematopoietic (CD45-) cells were characterized. Cell populations were isolated by flow cytometry for gene expression studies. 3,5-Diethoxycarbonyl-1,4-dihydrocollidine toxic injury induced cell cycling and expansion specifically in the subpopulation of oval cells in the periportal zone that express CD133. CD133+CD45- cells expressed hepatoblast and stem cell-associated genes, and single cells coexpressed both hepatocyte and cholangiocyte-associated genes, indicating bilineage potential. CD133+CD45- cells proliferated in response to liver injury. Following toxic hepatocyte damage, CD133+CD45- cells demonstrated upregulated expression of the hepatocyte gene Albumin. In contrast, toxic cholangiocyte injury resulted in upregulation of the cholangiocyte gene Ck19. After 21-28 days in culture, CD133+CD45- cells continued to generate cells of both hepatocyte and cholangiocyte lineages. Thus, CD133 expression identifies a population of oval cells in adult murine liver with the gene expression profile and function of primitive, bipotent liver stem cells. In response to lineage-specific injury, these cells demonstrate a lineage-appropriate genetic response. Disclosure of potential conflicts of interest is found at the end of this article.

Fluorescent immunohistochemistry and in situ hybridization analysis of mouse pancreas using low-power antigen-retrieval technique.

To facilitate the immunological reaction of antibodies with antigens in fixed tissues, it is necessary to unmask or retrieve the antigens through pretreatment of the specimens. However, adjustment of heating-induced antigen retrieval is always required for different tissues and antigens. In this study, by using a low-power antigen-retrieval technique with appropriate dilution of antibodies, we successfully immunostained key antigens in pancreas such as insulin, PDX-1, glucagon, cytokeratin, and CD31, which have previously presented a particular challenge for investigators because of the rapid autodigestion and high nonspecific antibody binding in this tissue. Satisfactory results were obtained when immunohistochemistry and fluorescence in situ hybridization analysis were combined in the same slides.

Fluorescent immunohistochemistry and in situ hybridization analysis of pancreas.

To facilitate the immunological reaction of antibodies with antigens in fixed tissues, it is necessary to unmask or retrieve the antigens through pretreatment of the specimens. However, adjustment of heat-induced antigen retrieval is always required for different tissues and antigens. Using a low-power antigen retrieval technique, with appropriate dilution of antibodies, we successfully immunostained key antigens in the pancreas such as insulin, PDX-1, glucagon, cytokeratin, and CD31, which have presented a particular challenge for investigators in the past, because of the rapid autodigestion and high nonspecific antibody binding in the tissue. Satisfactory results were obtained when immunohistochemistry and fluorescence in situ hybridization analysis were combined in the same slides.

Stable transgene expression in primitive human CD34+ hematopoietic stem/progenitor cells, using the Sleeping Beauty transposon system.

Sleeping Beauty (SB) transposon-mediated integration has been shown to achieve long-term transgene expression in a wide range of host cells. In this study, we improved the SB transposon-mediated gene transfer system for transduction of human CD34(+) stem/progenitor cells by two approaches: (1) to increase the transposition efficacy, a hyperactive mutant of SB, HSB, was used; (2) to improve the expression of the SB transposase and the transgene cassette carried by the transposon, different viral and cellular promoters were evaluated. SB components were delivered in trans into the target cells by Nucleoporation. The SB transposon-mediated integration efficacy was assessed by integrated transgene (enhanced green fluorescent protein [eGFP]) expression both in vitro and in vivo. In purified human cord blood CD34(+) cells, HSB achieved long-term transgene expression in nearly 7-fold more cells than the original SB transposase. Significantly brighter levels of eGFP expression (5-fold) were achieved with the human elongation factor 1alpha (EF1-alpha) promoter in Jurkat human T cells, compared with that achieved with the modified myeloproliferative sarcoma virus long terminal repeat enhancer-promoter (MNDU3); in contrast, the MNDU3 promoter expressed eGFP at the highest level in K-562 myeloid cells. In human CD34(+) cord blood cells studied under conditions directing myeloid differentiation, the highest transgene integration and expression were achieved using the EF1-alpha promoter to express the SB transposase combined with the MNDU3 promoter to express the eGFP reporter. Stable transgene expression was achieved at levels up to 27% for more than 4 weeks of culture after improved gene transfer to CD34(+) cells (average, 17%; n = 4). In vivo studies evaluating engraftment and differentiation of the SB-modified human CD34(+) cells demonstrated that SB-modified human CD34(+) cells engrafted in NOD/SCID/gamma chain(null) (NSG) mice and differentiated into multilineage cell types with eGFP expression. More importantly, secondary transplantation studies demonstrated that the integrated transgene was stably expressed in more primitive CD34(+) hematopoietic stem cells (HSCs) with long-term repopulating capability. This study demonstrates that an improved HSB gene transfer system can stably integrate genes into primitive human HSCs while maintaining the pluripotency of the stem cells, which shows promise for further advancement of non-virus-based gene therapy using hematopoietic stem cells.

Human intrathymic lineage commitment is marked by differential CD7 expression: identification of CD7- lympho-myeloid thymic progenitors.

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-lymphoid, and myeloid-erythroid conditions. Progressive restriction in lineage potential correlated with CD7 expression, that is, the CD7(hi) fraction produced T and NK cells but lacked B and myelo-erythroid potential, the CD7(int) (CD10(+)) fraction produced B, T, and NK cells, but lacked myelo-erythroid potential. The CD7(-) fraction produced all lymphoid and myelo-erythroid lineages and expressed HSC-associated genes. However, CD34(+)lin(-)CD7(-) thymocytes also expressed early T lymphoid genes Tdt, pTalpha, and IL-7Ralpha and lacked engraftment capacity, suggesting the signals that direct lymphoid commitment and corresponding loss of HSC function are rapidly initiated on arrival of HSC in the human thymus. Thus, differential levels of CD7 identify the progressive stages of lineage commitment in human thymus, initiated from a primitive CD7(-) lympho-myeloid thymic progenitor.

Expansion of multipotent and lymphoid-committed human progenitors through intracellular dimerization of Mpl.

Self-renewal capacity is rapidly lost during differentiation of hematopoietic stem cells to lineage-committed progenitors. We demonstrate here that regulated intracellular signaling through the cytokine receptor Mpl induces profound expansion of not only multipotent (ie, lymphomyeloid) but also lymphoid-committed human hematopoietic progenitors. A fusion protein containing the intracellular signaling domain of Mpl and a dimerization domain was constitutively expressed in populations enriched in human lymphomyeloid progenitor/stem cells (CD34(+)CD38(-)Lin(-)CD7(-)) and multilymphoid progenitors (CD34(+)CD38(-)Lin(-)CD7(+)). Intracellular dimerization of Mpl in target cells was induced by in vitro or in vivo administration of a diffusible synthetic ligand. In vitro, Mpl dimerization produced divisions of clonogenic, multilineage CD34(+) cells able to engraft immunodeficient mice. When dimerization was induced in vivo after transplantation of either lymphomyeloid or multilymphoid progenitors, donor-derived hematopoiesis was sustained for at least 12 weeks and primitive CD34(+)Lin(-) progenitors were expanded more than 1000-fold. Lineage potential of progenitors was not altered and differentiation was not prevented by synthetically induced Mpl signaling. These data demonstrate that dimerization of a single cytokine receptor can deliver a profound expansion signal in both uncommitted and lymphoid-committed human hematopoietic progenitors.

Bone marrow fails to differentiate into liver epithelium during murine development and regeneration.

UNLABELLED: Recent reports have provided conflicting conclusions regarding the role for bone marrow (BM)-derived cells in the regeneration of liver. Our aim was to investigate the potential of BM to contribute to liver epithelium using different BM transplant models designed to explore differentiation during normal liver development and regeneration after toxic injury. BM cells from transgenic green fluorescent protein (GFP) mice were injected into neonatal and adult immunodeficient and neonatal immune-competent mice. Three distinct models of liver injury were employed to test the contribution of marrow to the regeneration of hepatocytes, cholangiocytes, and oval cells in immune-deficient adult animals after neonatal transplant. Immunohistochemistry was combined with flow cytometry (FACS) and reverse transcription (RT)-PCR to increase the sensitivity and specificity of the analyses. Although GFP+ marrow-derived cells were observed in the livers of all transplanted animals, immunohistochemistry failed to demonstrate any marrow derived hepatocytes or cholangiocytes. FACS confirmed that GFP+ marrow-derived cells in the liver maintained expression of CD45, a leukocyte marker. Gene expression studies of GFP+ cells isolated by FACS failed to demonstrate expression of liver specific genes in these marrow-derived cells. CONCLUSION: Through highly sensitive and specific analyses, we were unable to demonstrate any evidence of transdifferentiation of BM-derived cells into epithelial hepatic tissue during the period of rapid growth in the neonatal period. Furthermore, although increased migration of hematopoietic cells to the liver occurred after toxic injury, these cells did not contribute directly to the replacement of hepatocytes, cholangiocytes, or oval cells.

Formation of pancreatic duct epithelium from bone marrow during neonatal development.

Recent reports suggest that bone marrow-derived cells engraft and differentiate into pancreatic tissue at very low frequency after pancreatic injury. All such studies have used adult recipients. The aim of our studies was to investigate the potential of bone marrow to contribute to the exocrine and endocrine components of the pancreas during the normal rapid growth of the organ that occurs during the neonatal period. Five to ten million bone marrow cells from adult, male, transgenic, green fluorescent protein (GFP) mice were injected into neonatal nonobese diabetic/severely compromised immunodeficient/beta2microglobulin-null mice 24 hours after birth. Two months after bone marrow transplantation, pancreas tissue was analyzed with fluorescence immunohistochemistry and fluorescence in situ hybridization (FISH). Co-staining of GFP, with anticytokeratin antibody, and with FISH for the presence of donor Y chromosome indicated that up to 40% of ducts (median 4.6%) contained epithelial cells derived from donor bone marrow. In some of these donor-derived ducts, there were clusters of large and small ducts, all comprised of GFP+ epithelium, suggesting that whole branching structures were derived from donor bone marrow. In addition, rare cells that coexpressed GFP and insulin were found within islets. Unlike pancreatic damage models, no bone marrow-derived vascular endothelial cells were found. In contrast to the neonatal recipients, bone marrow transplanted into adult mice rarely generated ductal epithelium or islet cells (p<.05 difference between adult and neonate transplants). These findings demonstrate the existence in bone marrow of pluripotent stem cells or epithelial precursors that can migrate to the pancreas and differentiate into complex organ-specific structures during the neonatal period.

Neonatal gene therapy of MPS I mice by intravenous injection of a lentiviral vector.

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.

Albumin-expressing hepatocyte-like cells develop in the livers of immune-deficient mice that received transplants of highly purified human hematopoietic stem cells.

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.