Human Stem Cell Biology Research Articles

Moving Forward in Human Mammary Stem Cell Biology and Breast Cancer Prognostication Using ALDH1

In this issue of Cell Stem Cell, the seminal work of Ginestier et al. (2007) elegantly demonstrates that aldehyde dehydrogenase 1 (ALDH1) facilitates the identification and isolation of normal and malignant human mammary stem cells in vitro, in vivo, and in situ in fixed tissues.

Bcl-XL modulates the differentiation of immortalized human neural stem cells

Understanding basic processes of human neural stem cell (hNSC) biology and differentiation is crucial for the development of cell replacement therapies. Bcl-X(L) has been reported to enhance dopaminergic neuron generation from hNSCs and mouse embryonic stem cells. In this work, we wanted to study, at the cellular level, the effects that Bcl-X(L) may exert on cell death during differentiation of hNSCs, and also on cell fate decisions and differentiation. To this end, we have used both v-myc immortalized (hNS1 cell line) and non-immortalized neurosphere cultures of hNSCs. In culture, using different experimental settings, we have consistently found that Bcl-X(L) enhances neuron generation while precluding glia generation. These effects do not arise from a glia-to-neuron shift (changes in fate decisions taken by precursors) or by only cell death counteraction, but, rather, data point to Bcl-X(L) increasing proliferation of neuronal progenitors, and inhibiting the differentiation of glial precursors. In vivo, after transplantation into the aged rat striatum, Bcl-X(L) overexpressing hNS1 cells generated more neurons and less glia than the control ones, confirming the results obtained in vitro. These results indicate an action of Bcl-X(L) modulating hNSCs differentiation, and may be thus important for the future development of cell therapy strategies for the diseased mammalian brain.

Neurodegeneration and cell replacement

The past decade has witnessed ground-breaking advances in human stem cell biology with scientists validating adult neurogenesis and establishing methods to isolate and propagate stem cell populations suitable for transplantation. These advances have forged promising strategies against human neurodegenerative diseases. For example, growth factor administration could stimulate intrinsic repair from endogenous neural stem cells, and cultured stem cells engineered into biopumps could be transplanted to deliver neuroprotective or restorative agents. Stem cells could also be transplanted to generate new neural elements that augment and potentially replace degenerating central nervous system (CNS) circuitry. Early efforts in neural tissue transplantation have shown that these strategies can improve functional outcome, but the ultimate success of clinical stem cell-based strategies will depend on detailed understanding of stem cell biology in the degenerating brain and detailed evaluation of their functional efficacy and safety in preclinical animal models.

"NeuroStem Chip": a novel highly specialized tool to study neural differentiation pathways in human stem cells

BackgroundHuman stem cells are viewed as a possible source of neurons for a cell-based therapy of neurodegenerative disorders, such as Parkinson's disease. Several protocols that generate different types of neurons from human stem cells (hSCs) have been developed. Nevertheless, the cellular mechanisms that underlie the development of neurons in vitro as they are subjected to the specific differentiation protocols are often poorly understood.ResultsWe have designed a focused DNA (oligonucleotide-based) large-scale microarray platform (named "NeuroStem Chip") and used it to study gene expression patterns in hSCs as they differentiate into neurons. We have selected genes that are relevant to cells (i) being stem cells, (ii) becoming neurons, and (iii) being neurons. The NeuroStem Chip has over 1,300 pre-selected gene targets and multiple controls spotted in quadruplicates (~46,000 spots total). In this study, we present the NeuroStem Chip in detail and describe the special advantages it offers to the fields of experimental neurology and stem cell biology. To illustrate the utility of NeuroStem Chip platform, we have characterized an undifferentiated population of pluripotent human embryonic stem cells (hESCs, cell line SA02). In addition, we have performed a comparative gene expression analysis of those cells versus a heterogeneous population of hESC-derived cells committed towards neuronal/dopaminergic differentiation pathway by co-culturing with PA6 stromal cells for 16 days and containing a few tyrosine hydroxylase-positive dopaminergic neurons.ConclusionWe characterized the gene expression profiles of undifferentiated and dopaminergic lineage-committed hESC-derived cells using a highly focused custom microarray platform (NeuroStem Chip) that can become an important research tool in human stem cell biology. We propose that the areas of application for NeuroStem microarray platform could be the following: (i) characterization of the expression of established, pre-selected gene targets in hSC lines, including newly derived ones, (ii) longitudinal quality control for maintained hSC populations, (iii) following gene expression changes during differentiation under defined cell culture conditions, and (iv) confirming the success of differentiation into specific neuronal subtypes.

The Leading Edge of Stem Cell Therapeutics

Stem cells, by virtue of their defining property of self-renewal, represent an unlimited source of potentially functional human cells for basic research and regenerative medicine. Having validated the feasibility of cell-based therapeutic strategies over the past decade, mostly through the use of rodent cells, the stem cell field has now embarked upon a detailed characterization of human cells. Recent progress has included improved cell culture conditions, long-term propagation, directed differentiation, and transplantation of both human embryonic and somatic stem cells. Continued progress in understanding basic human stem cell biology, combined with a better handle on the fundamental pathophysiology of human diseases one wishes to target (including the use of human stem cells in primate and other large animal models of human disease), should help to move this technology closer to clinical application.

Knockdown of the Fanconi Anemia Gene FANCD2 Directly Affects Hematopoiesis in Human Embryonic Stem Cells.

Fanconi anemia (FA) is a rare autosomal recessive disease characterized by the progressive bone marrow failure, developmental anomalies and cancer susceptibility. Twelve distinct FA complementation groups have been identified (A–C, D1, D2, E–G, I, J, L, M) and 11 cDNAs cloned. A current working model proposes that eight FA proteins (A, B, C, E, F, G, L and M) assemble to form a multi-protein nuclear complex, involved the repair of damaged DNA. The FA complex facilitates the monoubiquitination of the FANCD2 protein following exposure to DNA damaging agents. Ubiquinated FANCD2 co-localizes with the key DNA repair proteins RAD51, BRCA2 (know as FANCD1), BRCA1, and FANCJ (BACH1) to promote homologous recombination. The hallmark of FA patients is marrow failure due to defective hematopoietic stem cells. The study of human FA stem cell biology is severely restricted due to the few CD34+ cells that can be isolated from FA patients compared with normal individuals. We generated human embryonic stem cells (hESC) exhibiting FA phenotype by introducing small double strand RNA species to ablate FANCD2 gene function in hESC. Human ES cells were then differentiated into hematopoietic cells to investigate FA hematopoiesis. FANCD2-specific small hairpin RNAs (shRNAs) were designed and cloned into a self-inactivated lentiviral vector with a GFP cDNA. High titer virus (2×108 iu/ml) was used for transduction of the hESC line H1. More than 90% of H1 cells were transduced with lentivirus that was observed by GFP expression. The FANCD2 protein expression was analyzed by Western blotting; FANCD2 shRNA targeted cells expressed 1–3% of the FANCD2 protein compared with control H1 cells. Functional assessment of the H1-FANCD2 was performed by incubation with DNA cross-linking agent, mitomycin C (MMC). The H1-FANCD2 cells were nearly 10 times more sensitive to MMC than untreated or scrambled shRNA ES cells. The effective dose 50 (ED50) of MMC required to induce apoptosis was only 4 ng/ml in H1-FANCD2 cells compared with 30 ng/ml in mock-infected of scrambled shRNA controls. Cytogentic abnormalities are the hallmark of Fanconi anemia. When H1-FANCD2 cells were exposed to MMC, 57% of cells had abnormal cytogenetics compared with 2% of scrambled shRNA transduced H1 cells. In addition, 75.34±0.69% of H1-FANCD2 cells were arrested at the G2/M phase of the cell cycle after MMC exposure compared with 60.63±2.5% of H1-scramble cells. MMC hypersensitivity, cell cycle defects and chromosomal abnormalities of H1-FANCD2 confirmed the FA phenotype. Differentiation of H1 and H1-scramble cells through a blastocyst intermediate produced a discrete number of KDR+/CD31+ hemangioblast cells that give rise to erythroid and myeloid hematopoietic colonies in methylcellulose culture. H1-FANCD2 ES cells produced blastocyst and hemangioblast cells without significant development of end-stage hematopoietic lineages. In conclusion, we successfully converted, using shRNAs, hESC H1 cells that exhibit the FA phenotype including abnormal hematopoiesis. Generation of mutant FA hES cells will be a valuable model to study FA pathophysiology and treatment.

Human CD34+ cells expressing the inv(16) fusion protein exhibit a myelomonocytic phenotype with greatly enhanced proliferative ability

The t(16:16) and inv(16) are associated with FAB M4Eo myeloid leukemias and result in fusion of the CBFB gene to the MYH11 gene (encoding smooth muscle myosin heavy chain [SMMHC]). Knockout of CBFbeta causes embryonic lethality due to lack of definitive hematopoiesis. Although knock-in of CBFB-MYH11 is not sufficient to cause disease, expression increases the incidence of leukemia when combined with cooperating events. Although mouse models are valuable tools in the study of leukemogenesis, little is known about the contribution of CBFbeta-SMMHC to human hematopoietic stem and progenitor cell self-renewal. We introduced the CBFbeta-MYH11 cDNA into human CD34+ cells via retroviral transduction. Transduced cells displayed an initial repression of progenitor activity but eventually dominated the culture, resulting in the proliferation of clonal populations for up to 7 months. Long-term cultures displayed a myelomonocytic morphology while retaining multilineage progenitor activity and engraftment in NOD/SCID-B2M-/- mice. Progenitor cells from long-term cultures showed altered expression of genes defining inv(16) identified in microarray studies of human patient samples. This system will be useful in examining the effects of CBFbeta-SMMHC on gene expression in the human preleukemic cell, in characterizing the effect of this oncogene on human stem cell biology, and in defining its contribution to the development of leukemia.

Characterization and Gene Expression Profiling of Five New Human Embryonic Stem Cell Lines Derived in Taiwan

Many human embryonic stem cell (hESC) lines have been reported, but only a few of them have been fully characterized. In this report, five new hESC lines were derived from 32 discarded blastocysts in Taiwan, and these lines were continuously cultured on mitotically inactivated mouse embryonic fibroblast (MEF) feeder layer in the hESC medium for more than 44 passages and underwent freezing/thawing processes. All five hESC lines expressed characteristic undifferentiated hESC markers, such as SSEA-4, TRA-1-81, alkaline phosphatase, TERT, and the transcription factors POU5F1 (OCT4) and NANOG. hESC lines T1 and T3 possess normal female karyotypes, whereas lines T4 and T5 are normal male, but line T2 is male trisomy 12 (47XY,+12). hESC lines T1, T2, T3, and T5 were able to produce teratomas in severe combined immunodeficient (SCID) mice, and line T4 could only form embryoid bodies (EBs) in vitro. Global gene expression profiles of these five newly derived hESC lines were analyzed using the Affymetrix human genome U133 plus 2.0 GeneChip. The results showed that 4,145 transcripts, including 19% of unknown functions, were detected in all five hESC lines. Comparison of the 4,145 genes commonly expressed in the five hESC lines with those genes expressed in teratomas produced by the hESC line T1 and placenta revealed 40 genes exclusively expressed in all five hESC lines. These 40 genes include the previously reported stemness genes, such as POU5F1 (OCT4), NANOG, TDGF1 (CRIPTO), SALL4, LECT1, and BUB1 responsible for self-renewal and pluripotent differentiation. The global gene expression analysis also indicated that the transforming growth factor-beta (TGF-beta)/activin branch components inhibin BC, ACVR2A, ACVR1 (ALK2), TGFBR1 (ALK5), and SMAD2 were found to be highly expressed in undifferentiated states of these five hESC lines and decreased upon differentiation. In short, the hESC nature of these five hESC lines is supported by the undifferentiated state, extensive renewal capacity, and pluripotency, including the ability to form teratomas and/or EBs. These cell lines will be useful for human embryonic stem cell biology and drug development.

365. Human Embryonic Stem Cells: Genetic Conversion to the Fanconi Anemia Phenotype

Fanconi anemia (FA) is a rare autosomal recessive disease characterized by the progressive bone marrow failure, developmental anomalies and cancer susceptibility. Twelve distinct FA complementation groups have been identified (A|[ndash]|C, D1, D2, E|[ndash]|G, I, J, L, M) and 11 cDNAs cloned. A current working model proposes that eight FA proteins (A, B, C, E, F, G, L and M) assemble to form a multi-protein nuclear complex, involved the repair of damaged DNA. The FA complex facilitates the monoubiquitination of the FANCD2 protein following exposure to DNA damaging agents. Ubiquinated FANCD2 co-localizes with the key DNA repair proteins RAD51, BRCA2 (know as FANCD1), BRCA1, and FANCJ (BACH1) to promote homologous recombination. The hallmark of FA patients is marrow failure due to defective hematopoietic stem cells. The study of human FA stem cell biology is severely restricted due to the few CD34+ cells that can be isolated from FA patients compared with normal individuals. We set out to create a reusable source of human FA hematopoietic cells by introducing small double strand RNA species to ablate specific FA gene function in human embryonic stem cells (hESC). Human ES cells can then differentiate into human CD34+ cells to investigate human FA hematopoiesis. FANCD2-specific small hairpin RNAs (shRNAs) were designed and cloned into a self-inactivated lentiviral vector with a GFP cDNA. High titer virus (2|[times]|108 iu/ml) was used for transduction of the hESC line H1. FACS sorted hESC-FANCD2 clones were tested. FANCD2 protein expression was analyzed by Western blotting; FANCD2 shRNA targeted cells expressed 1|[ndash]|2% of the FANCD2 protein compared with control (mock transduced or scrambled shRNA) H1 cells. Functional assessment of the hESC-FANCD2 was performed by incubation with DNA cross-linking agent, mitomycin C (MMC). The hESC-FANCD2 cells were nearly 20 times more sensitive to MMC than untreated or scrambled shRNA ES cells. The effective dose 50 (ED50) of MMC required to induce apoptosis was only 5 ng/ml in hESC-FANCD2 cells compared with 80 ng/ml in mock- infected of scrambled shRNA controls. The expected cytogenetic aberrations were identified in hESC-FAND2 but not control cells confirming the FA phenotype. Differentiation of hESC-FANCD2 cells through a blastocyst intermediate produced a small but discrete number of CD34+/c-kit+ cells that form hematopoietic colonies in methylcellulose culture. In conclusion, we have successfully converted, using shRNAs, hESC H1 cells into cells exhibit the FA phenotype. Generation of hESC-FANCD2 cells provides a ready source of multipotent hematopoietic progenitors and a powerful tool for investigation of FA hematopoiesis. To our knowledge, this is the first description of the genetic conversion of hESCs into a disease phenotype.

Biology of Normal and Acute Myeloid Leukemia Stem Cells

The substantial understanding that has been gained over the past 5 decades of the biology of blood formation is largely due to the development of functional quantitative assays for cells at all stages of differentiation, from multipotential stem cells to mature cells. The majority of studies have involved the mouse because the ease with which repopulation studies can be carried out with this animal model allows the assay of complete lineage development from stem cells. In the past decade, advances in repopulation assays for human stem cells using xenotransplantation have greatly enhanced our understanding of human stem cell biology. Importantly, the xenotransplantation methodology has also been used to identify the cancer stem cell that initiates and sustains leukemic proliferation, providing key evidence for the cancer stem cell hypothesis. This hypothesis argues that cancer cells are functionally heterogeneous and hierarchically organized such that only specific cells are capable of sustaining tumor growth and continuously producing the cells that make up the bulk of the tumor. Recent studies have also brought into focus the importance of the intimate relationship between the stem cell (normal or leukemic) and its microenvironment. Coming into view are the molecular players involved in stem cell homing, migration, and adhesion, as well as the cellular components of the microenvironmental niche. Here we review recent studies that have begun, to elucidate the interplay between normal and leukemic human stem cells and their microenvironment.

Human CD34+ Cells Expressing CBFβ-MYH11 Exhibit a Myelomonocytic Phenotype with Greatly Enhanced Proliferative Ability.

The Core Binding Factor (CBF) transcription factor complex is required for normal hematopoiesis. Knockout of either component of the complex, AML1 or CBFβ, results in embryonic lethality in mice due to a lack of definitive hematopoiesis. Chromosomal rearrangements involving the CBF genes are commonly associated with leukemia. The t(8:21) fuses the AML1 gene to ETO (AE) and is predominantly observed in leukemias of the FAB M2 subtype of acute myeloid leukemia (AML). The t(16:16) and inv(16) are associated with FAB M4Eo leukemias and result in fusion of the CBFβ gene to MYH11 (CM). While knockin of either AE or CM is not sufficient to cause disease, expression of either protein increases the incidence of leukemia in mice when combined with mutagens or specific secondary genetic manipulations. We have previously shown that retroviral transduction of primary human CD34+ cells with AE causes a dramatic accumulation of CD34+ progenitor cells with increased CFU-C and CAFC activity during an increased lifespan in vitro. These cultures retain the ability to differentiate to myeloid, lymphoid, and erythroid lineages and the ability to engraft NOD/SCID mice. Consistent with mouse studies, these cells fail to cause disease in mice or become transformed in vitro. These cultures serve as a preleukemic model for human AML associated with AML1-ETO expression. We show here that retroviral transduction of CM into human CD34+ hematopoietic cells similarly mimics a putative pre-leukemic condition initiated by inv(16). Transduced cells proliferate for up to 7 months in culture with retention of CD34 expression on a subset of cells. Cells from CM cultures display a more mature morphology than AE cells, consistent with the association with M4Eo AMLs, and have a greatly reduced ability to differentiate to the erythroid lineage in methylcellulose assays. Nevertheless, long-term CM cultures retain CFU-C activity, the ability to engraft NOD/SCID-B2M mice, and the potential for B-cell development in vitro, implicating a primitive progenitor as the target cell rather than a committed granulocyte/monocyte progenitor. Microarray analysis of AML leukemias has identified specific genes with altered expression in inv(16) patient samples. We have found that a number of these inv(16)-associated transcripts are likewise altered in CD34+ cells from our CM cultures when compared to control CD34+ cells, while others are not affected. These data suggest that our system will be useful in examining the effects of CBFβ-MYH11 on gene expression in the human preleukemic cell, in characterizing the effect of this oncogene on human stem cell biology and in defining its contribution to the development of leukemia.

Differential Expression Profiling of Membrane Proteins by Quantitative Proteomics in a Human Mesenchymal Stem Cell Line Undergoing Osteoblast Differentiation

One of the major limitations for understanding the biology of human mesenchymal stem cells (hMSCs) is the absence of prospective markers needed for distinguishing them from other cells and for monitoring lineage-specific differentiation. Mass spectrometry (MS)-based proteomics has proven extremely useful for analyzing complex protein expression patterns and, when applied quantitatively, can be used to resolve subtle differences between samples. Thus, we used MS to characterize changes in expression of membrane protein markers before and after short-term induction of osteoblast (OB) differentiation in a cell model of hMSCs established by overexpression of human telomerase reverse-transcriptase gene. We identified 463 unique proteins with extremely high confidence, including all known markers of hMSCs (e.g., SH3 [CD71], SH2 [CD105], CD166, CD44, Thy1, CD29, and HOP26 [CD63]) among 148 integral membrane or membrane-anchored proteins and 159 membrane-associated proteins. Twenty-nine integrins and cell adhesion molecules, 20 receptors, and 18 Ras-related small GTPases were also identified. Upon OB differentiation, the expression levels of 83 proteins increased by at least twofold whereas the levels of another 21 decreased by at least twofold. For example, alkaline phosphatase (ALP), versican core protein, and tenascin increased 27-, 12-, and 4-fold, respectively, and fatty acid synthase decreased sixfold. The observed increases in veriscan and ALP were confirmed using immunocytochemistry and cytochemistry. Quantitative real-time reverse transcription-polymerase chain reaction confirmed the presence of mRNA of these membrane proteins. However, with the exception of ALP, no concordance was detected between the changes in levels of gene and protein expression during OB differentiation. In conclusion, MS-based proteomics can reveal novel markers for MSCs that can be used for their isolation and for monitoring OB differentiation.

Human Blastocyst Culture and Derivation of Embryonic Stem Cell Lines

Human embryonic stem cell (hESC) biology is expected to revolutionize the future of medicine by the provision of cell-based therapies for the treatment of a variety of deliberatig diseases. The tremendous versatility of hESCs has reinforced this hope. To understand the biology of these mysterious cells and attempt to differentiate them into desirable tissues, bona fide hESCs that maintain their stability with time are required for research and clinical application. This review discusses the various protocols to derive and propagate hESCs from high quality embryos. The nature and properties of hESCs are also described together with unanswered questions that need to be addressed if this science is to be taken to the bedside.

Issues in Human Embryonic Stem Cell Biology

Human embryonic stem cells (hESCs) derived from frozen unused blastocytes from in vitro fertilization clinics exhibit unlimited proliferative capacity, and are pluripotent. The ability to maintain hESCs cultures in an undifferentiated state in vitro and differentiate them along a given lineage in a controlled manner make these cells potentially invaluable in regenerative medicine. Recent developments in the generation of patient-specific hESCs as well as animal product-free culture systems are some of the latest developments in the field; however great challenges lie ahead before the therapeutic application of hESCs becomes a reality. This article addresses some of the successes and roadblocks concerning the hESCs field; including various political, ethical and scientific obstacles.

Model systems in stem cell biology.

Stem cell scientists and ethicists have focused intently on questions relevant to the developmental stage and developmental capacities of stem cells. Comparably less attention has been paid to an equally important set of questions about the nature of stem cells, their common characteristics, their non-negligible differences and their possible developmental species specificity. Answers to these questions are essential to the project of justly inferring anything about human stem cell biology from studies in non-human model systems--and so to the possibility of eventually developing human therapies based on stem cell biology. After introducing and discussing these questions, I conclude with a brief discussion of the creation of novel model systems in stem cell biology: human-to-animal embryonic chimeras. Such novel model systems may help to overcome obstacles to extrapolation, but they are also scientifically and ethically contentious.

Telomere biology of human hematopoietic stem cells.

Telomeres are protein DNA structures present at the ends of chromosomes and are essential for genetic stability and cell replication. Telomerase is the enzyme complex that maintains telomere integrity. Hematopoietic stem cells express telomerase and contain long telomeres, which become shorter as cells differentiate and mature. The extent of telomere shortening and the level of telomerase activity often correlate with the presence and severity of some hematopoietic diseases. The fundamentals of telomeres and telomerase are reviewed, and the telomere biology of human hematopoietic cells is discussed. Telomere length and telomerase activity are important in the self-renewal of hematopoietic stem cells. Changes within these compartments affect both normal hematopoietic cells and the generation of hematopoietic disease. Telomere length provides information pertaining to the proliferative history and potential of a hematopoietic cell. The role of telomerase and telomeres within the hematopoietic compartment needs further clarification. Advances in our knowledge in this field may improve clinical outcomes for the treatment of hematologic disease.

Effects of Wnt signaling on proliferation and differentiation of human mesenchymal stem cells.

Mesenchymal stem cells are pluripotent cells from bone marrow, which can be differentiated into the osteogenic, chondrogenic, and adipogenic lineages in vitro and are a source of cells in bone and cartilage tissue engineering. An improvement in current tissue-engineering protocols requires more detailed insight into the molecular cues that regulate the distinct steps of osteochondral differentiation. Because Wnt signaling has been widely implicated in mesenchymal differentiation, we analyzed the role of Wnt signaling in human mesenchymal stem cell (hMSC) biology by stimulation of the pathway with lithium chloride and Wnt3A-conditioned medium. We demonstrate a role for low levels of Wnt signaling in proliferation of uncommitted hMSCs and confirm that Wnt signaling controls osteoprogenitor proliferation. On the other hand, at high Wnt levels we observed a block in adipogenic differentiation and an increase in the expression of alkaline phosphatase, suggesting a role in the initiation of osteogenesis. The results of this study suggest that bone tissue engineering could benefit from the activation of critical levels of Wnt signaling at defined stages of differentiation. Moreover, our data suggest that hMSCs provide a valid in vitro model to study the role of Wnt signaling in mesenchymal biology.

Immune-deficient mouse models for analysis of human stem cells.

The field of murine models of xenotransplantation has grown immensely over the past two decades. The explosive growth in this field is in part due to the fact that good in vitro methods do not exist yet to allow examination of human stem cell homing into the bone marrow compartment versus other tissues, long-term survival of human stem cells, or differentiation into tissues outside of the hematopoietic system. Since these important aspects of human stem cell biology can be examined in vivo using immune-deficient mice, the number of different strains and models is constantly increasing. The current review discusses the merits and drawbacks of each immune-deficient mouse xenograft system as it stands to date and reviews how each immune-deficient mouse model has been used to further our knowledge of human hematopoietic stem cell biology.

C1qRp defines a new human stem cell population with hematopoietic and hepatic potential.

The characterization of two distinct classes of hematopoietic stem cells based on CD34 expression and the ability of human bone marrow (BM) cells to differentiate into nonhematopoietic cells introduced new levels of complexity within the stem cell compartment. Here we report the identification and purification of a rare human stem cell population with hematopoietic and hepatic potential based on the expression of a receptor for the complement molecule C1q (C1qR(p)). We show that C1qR(p) is a positive marker of all BM-repopulating stem cells because it is expressed on both CD34(-) and CD34(+) stem cells from umbilical cord blood and adult BM. In addition, we show that highly purified lineage-negative CD45(+)CD38(-)CD34(+or-)C1qR(p)(+) cells not only have BM-repopulating capacity but also can differentiate into human hepatocytes in vivo. The identification of human hepatocytes in mouse livers indicates that the NOD/SCID (nonobese diabetic/severe combined immunodeficient) mouse model can be a valuable tool to study the differentiation potential of adult human stem cells. These findings may have important scientific and clinical implications in the field of human stem cell biology and transplantation.

Overview of the Current Status of Research on Human Pluripotent Stem Cells

Seventeen years after the first description of the derivation and properties of mouse embryonic stem (ES) cells [1, 2], Thomson and co-workers [3] reported the isolation of human ES cells from blastocysts. The derivation of human ES cells has led to hopes that their availability will revolutionise biomedical research and transplantation medicine. While it is not difficult to envision potential applications of human ES cells in research or regenerative medicine, many obstacles must be overcome before these cells are used, for example, in high throughput assays to elucidate gene function or to screen chemical libraries for active compounds, and before clinical trials in transplantation can begin. Although the challenges are complex, there has already been significant progress in our basic understanding of human pluripotent stem cell biology. I review that progress here. I consider aspects of the biology of human ES cells and compare them to the better characterised mouse ES cells, then discuss recent advances in the biology of human ES cells. This review will also touch on the more limited studies to date using embryonic germ (EG cells) [4] derived from human embryonic or foetal gonads.