Saul J. Sharkis

Multi-Organ, Multi-Lineage Engraftment by a Single Bone Marrow-Derived Stem Cell

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.

Hematopoietic stem cells convert into liver cells within days without fusion.

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.

Activin A Maintains Self-Renewal and Regulates Fibroblast Growth Factor, Wnt, and Bone Morphogenic Protein Pathways in Human Embryonic Stem Cells

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 self‐renewal 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.

Generation of endoderm‐derived human induced pluripotent stem cells from primary hepatocytes

Recent advances in induced pluripotent stem (iPS) cell research have significantly changed our perspective on regenerative medicine. Patient‐specific iPS cells have been derived not only for disease modeling but also as sources for cell replacement therapy. However, there have been insufficient data to prove that iPS cells are functionally equivalent to human embryonic stem (hES) cells or are safer than hES cells. There are several important issues that need to be addressed, and foremost are the safety and efficacy of human iPS cells of different origins. Human iPS cells have been derived mostly from cells originating from mesoderm and in a few cases from ectoderm. So far, there has been no report of endoderm–derived human iPS cells, and this has prevented comprehensive comparative investigations of the quality of human iPS cells of different origins. Here we show for the first time reprogramming of human endoderm‐derived cells (i.e., primary hepatocytes) to pluripotency. Hepatocyte‐derived iPS cells appear indistinguishable from hES cells with respect to colony morphology, growth properties, expression of pluripotency‐associated transcription factors and surface markers, and differentiation potential in embryoid body formation and teratoma assays. In addition, these cells are able to directly differentiate into definitive endoderm, hepatic progenitors, and mature hepatocytes. Conclusion: The technology to develop endoderm–derived human iPS cell lines, together with other established cell lines, will provide a foundation for elucidating the mechanisms of cellular reprogramming and for studying the safety and efficacy of differentially originated human iPS cells for cell therapy. For the study of liver disease pathogenesis, this technology also provides a potentially more amenable system for generating liver disease‐specific iPS cells. (HEPATOLOGY 2010;51:1810–1819)

In Vivo Liver Regeneration Potential of Human Induced Pluripotent Stem Cells from Diverse Origins

Hepatic cells derived from human induced pluripotent stem cells of various origins contribute to liver regeneration in vivo. Treating Liver Disease, A Promethean Task As the ancient Greek legend of the disgraced Prometheus showed, the only human organ that can regenerate itself is the liver. Despite the liver’s remarkable capacity for repair and regeneration, diseases such as liver cirrhosis or hepatocellular carcinoma eventually destroy this ability and the only option is for patients to receive a liver transplant. But there is a severe shortage of donor livers for transplantation, which has prompted interest in stem cell therapy for treating patients with end-stage liver disease. However, liver stem cells are difficult to isolate and expand in culture so alternatives are being sought. Enter Liu et al. with a stem cell strategy that involves deriving mature human liver cells (hepatocytes) from human induced pluripotent stem cells (iPSCs). First, these investigators generated human iPSCs from a variety of adult human cells including hepatocytes, fibroblasts, and keratinocytes and showed that although these iPSCs were derived from very different cell types, they retained similar (although not identical) epigenetic signatures. The authors then used an established stepwise differentiation protocol to induce these human iPSCs to differentiate along the hepatic lineage first into definitive endoderm, then hepatic progenitor cells, and finally into mature hepatocyte-like cells. They found that, regardless of their origin, the different human iPSC lines all showed the same ability to differentiate into hepatic cells. To be useful for cell therapy, these human iPSC-derived hepatic cells must be able to engraft in liver tissue and function in the same way as normal human hepatocytes. So, the authors tested their human iPSC-derived hepatic cells (at different stages of differentiation) for their ability to engraft liver tissue in a xenograft model comprising immunodeficient mice treated with a chemical to induce liver injury. They intravenously infused the mice with 2 million human iPSC-derived hepatic cells or with normal human hepatocytes as a control. They found that human iPSC-derived hepatic cells engrafted mouse liver with an efficiency ranging from 8 to 15%, comparable to that for adult human hepatocytes (~11%). But were the engrafted human hepatic cells functional? The authors report that proteins normally secreted by adult human hepatocytes, such as albumin, transferrin, α-1-antitrypsin, and fibrinogen, could be detected in the serum of mice transplanted with human iPSC-derived hepatic cells at concentrations of 46, 101, 8.1, and 1100 ng/ml, respectively. Although preliminary, these encouraging findings suggest that it may be possible in the future to use infusions of human iPSC-derived hepatic cells to rescue injured liver tissue in patients with end-stage liver disease. 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.

A clinically relevant population of leukemic CD34+CD38- cells in acute myeloid leukemia

Relapse of acute myeloid leukemia (AML) is thought to reflect the failure of current therapies to adequately target leukemia stem cells (LSCs), the rare, resistant cells presumed responsible for maintenance of the leukemia and typically enriched in the CD34(+)CD38(-) cell population. Despite the considerable research on LSCs over the past 2 decades, the clinical significance of these cells remains uncertain. However, if clinically relevant, it is expected that LSCs would be enriched in minimal residual disease and predictive of relapse. CD34(+) subpopulations from AML patients were analyzed by flow cytometry throughout treatment. Sorted cell populations were analyzed by fluorescence in situ hybridization for leukemia-specific cytogenetic abnormalities (when present) and by transplantation into immunodeficient mice to determine self-renewal capacity. Intermediate (int) levels of aldehyde dehydrogenase (ALDH) activity reliably distinguished leukemic CD34(+)CD38(-) cells capable of engrafting immunodeficient mice from residual normal hematopoietic stem cells that exhibited relatively higher ALDH activity. Minimal residual disease detected during complete remission was enriched for the CD34(+)CD38(-)ALDH(int) leukemic cells, and the presence of these cells after therapy highly correlated with subsequent clinical relapse. ALDH activity appears to distinguish normal from leukemic CD34(+)CD38(-) cells and identifies those AML cells associated with relapse.

Functional activity of murine CD34+and CD34− hematopoietic stem cell populations

The transmembrane glycoprotein CD34 is expressed on human hematopoietic stem cells and committed progenitors in the bone marrow, and CD34-positive selection currently is used to isolate bone marrow repopulating cells in clinical transplantation protocols. Recently, CD34- hematopoietic stem cells were described in both humans and mice, and it was suggested that CD34+ murine bone marrow cells may lack long-term reconstituting ability. In this study, the long-term repopulating ability of CD34+Lin- vs CD34-Lin- cells was compared directly using syngeneic murine bone marrow transplantation. Highly purified populations of CD34+Lin- and CD34-Lin- cells each are able to reconstitute bone marrow, confirming that both populations contain hematopoietic stem cells; however, the number of hematopoietic stem cells in the CD34+Lin- fraction is approximately 100-fold greater than the number in the CD34-Lin- fraction. In competitive repopulation experiments, CD34+ stem cells are better able to engraft the bone marrow than are CD34- cells. CD34+Lin- cells provide both short- and long-term engraftment, but the CD34-Lin- cells are capable of only long-term engraftment. Ex vivo, the CD34+Lin- stem cells expand over 3 days in culture and maintain the ability to durably engraft animals in a serial transplant model. In contrast, when CD34-Lin- cells are cultured using the same conditions ex vivo, the cell number decreases, and the cells do not retain the ability to repopulate the bone marrow. Thus, the CD34+Lin- and CD34-Lin- cells constitute two functionally distinct populations that are capable of long-term bone marrow reconstitution.

Reprogramming of EBV-immortalized B-lymphocyte cell lines into induced pluripotent stem cells

EBV-immortalized B lymphocyte cell lines have been widely banked for studying a variety of diseases, including rare genetic disorders. These cell lines represent an important resource for disease modeling with the induced pluripotent stem cell (iPSC) technology. Here we report the generation of iPSCs from EBV-immortalized B-cell lines derived from multiple inherited disease patients via a nonviral method. The reprogramming method for the EBV cell lines involves a distinct protocol compared with that of patient fibroblasts. The B-cell line-derived iPSCs expressed pluripotency markers, retained the inherited mutation and the parental V(D)J rearrangement profile, and differentiated into all 3 germ layer cell types. There was no integration of the reprogramming-related transgenes or the EBV-associated genes in these iPSCs. The ability to reprogram the widely banked patient B-cell lines will offer an unprecedented opportunity to generate human disease models and provide novel drug therapies.

Characterization of chronic myeloid leukemia stem cells.

Although tyrosine kinase inhibitors have redefined the care of chronic myeloid leukemia (CML), these agents have not proved curative, likely due to resistance of the leukemia stem cells (LSC). While a number of potential therapeutic targets have emerged in CML, their expression in the LSC remains largely unknown. We therefore isolated subsets of CD34+ stem/progenitor cells from normal donors and from patients with chronic phase or blast crisis CML. These cell subsets were then characterized based on ability to engraft immunodeficient mice and expression of candidate therapeutic targets. The CD34+CD38− CML cell population with high aldehyde dehydrogenase (ALDH) activity was the most enriched for immunodeficient mouse engrafting capacity. The putative targets: PROTEINASE 3, SURVIVIN, and hTERT were expressed only at relatively low levels by the CD34+CD38−ALDHhigh CML cells, similar to the normal CD34+CD38−ALDHhigh cells and less than in the total CML CD34+ cells. In fact, the highest expression of these antigens was in normal, unfractionated CD34+ cells. In contrast, PRAME and WT1 were more highly expressed by all CML CD34+ subsets than their normal counterparts. Thus, ALDH activity appears to enrich for CML stem cells, which display an expression profile that is distinct from normal stem/progenitor cells and even the CML progenitors. Indeed, expression of a putative target by the total CD34+ population in CML does not guarantee expression by the LSC. These expression patterns suggest that PROTEINASE 3, SURVIVIN, and hTERT are not optimal therapeutic targets in CML stem cells; whereas PRAME and WT1 seem promising. Am. J. Hematol., 2011.

Dye-mediated photolysis of normal and neoplastic hematopoietic cells.

The purpose of this study was to determine the sensitivity to merocyanine 540 (MC 540)-mediated photolysis of normal human hematopoietic progenitor cells and four leukemia cell lines (Daudi, Raji, K562 and HL-60). Late erythroid progenitors were the most sensitive normal cells. Early erythroid progenitors were of intermediate sensitivity. Granulocyte/macrophage progenitors and multipotent progenitors were the least sensitive normal marrow cells. A combination of dye concentration, serum concentration, and illumination that eliminated 50% of multipotent progenitor cells reduced the concentration of leukemic cells by greater than or equal to 4.5 log. It is conceivable that this difference in photosensitivity can be exploited for the extracorporeal purging of autologous remission marrow grafts.