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Directed differentiation of human induced pluripotent stem cells (iPSCs) toward hepatobiliary lineages has been increasingly used as models of human liver development/diseases. As protein kinases are important components of signaling pathways regulating cell fate changes, we sought to define the key molecular mediators regulating human liver development using inhibitors targeting tyrosine kinases during hepatic differentiation of human iPSCs. A library of tyrosine kinase inhibitors was used for initial screening during the multistage differentiation of human iPSCs to hepatic lineage. Among the 80 kinase inhibitors tested, only Src inhibitors suppressed endoderm formation while none had significant effect on later stages of hepatic differentiation. Transient inhibition of c-Src during endodermal induction of human iPSCs reduced endodermal commitment and expression of endodermal markers, including SOX17 and FOXA2, in a dose-dependent manner. Interestingly, the transiently treated cells later developed into profibrogenic cholangiocyte-like cells expressing both cholangiocyte markers, such as CK7 and CK19, and fibrosis markers, including Collagen1 and smooth muscle actin. Further analysis of these cells revealed colocalized expression of collagen and yes-associated protein (YAP; a marker associated with bile duct proliferation/fibrosis) and an increased production of interleukin-6 and tumor necrosis factor-α. Moreover, treatment with verteporfin, a YAP inhibitor, significantly reduced expression of fibrosis markers. In summary, these results suggest that c-Src has a critical role in cell fate determination during endodermal commitment of human iPSCs, and its alteration in early liver development in human may lead to increased production of abnormal YAP expressing profibrogenic proinflammatory cholangiocytes, similar to those seen in livers of patients with biliary fibrosis. Stem Cells 2019;37:306-317., (© AlphaMed Press 2018.)

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.

To maintain the integrity of the organism, embryonic stem cells (ESC) need to maintain their genomic integrity in response to DNA damage. DNA double strand breaks (DSBs) are one of the most lethal forms of DNA damage and can have disastrous consequences if not repaired correctly, leading to cell death, genomic instability and cancer. How human ESC (hESC) maintain genomic integrity in response to agents that cause DSBs is relatively unclear. Adult somatic cells can be induced to "dedifferentiate" into induced pluripotent stem cells (iPSC) and reprogram into cells of all three germ layers. Whether iPSC have reprogrammed the DNA damage response is a critical question in regenerative medicine. Here, we show that hESC demonstrate high levels of endogenous reactive oxygen species (ROS) which can contribute to DNA damage and may arise from high levels of metabolic activity. To potentially counter genomic instability caused by DNA damage, we find that hESC employ two strategies: First, these cells have enhanced levels of DNA repair proteins, including those involved in repair of DSBs, and they demonstrate elevated nonhomologous end-joining (NHEJ) activity and repair efficacy, one of the main pathways for repairing DSBs. Second, they are hypersensitive to DNA damaging agents, as evidenced by a high level of apoptosis upon irradiation. Importantly, iPSC, unlike the parent cells they are derived from, mimic hESC in their ROS levels, cell cycle profiles, repair protein expression and NHEJ repair efficacy, indicating reprogramming of the DNA repair pathways. Human iPSC however show a partial apoptotic response to irradiation, compared to hESC. We suggest that DNA damage responses may constitute important markers for the efficacy of iPSC reprogramming., (2011 Elsevier B.V. All rights reserved.)

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.

Unlabelled: 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.

Using an in vivo selection technique, we can isolate individual cells that can repopulate the hematopoietic system of a lethally irradiated murine recipient. These cells rapidly acquire a CD34 phenotype in the animal. Progenitors in our long-term chimeras are of donor type. We also have evidence that transplantation of limiting numbers (as low as a single cell) that have this long-term repopulating ability (LTRA) can self-renew. This is demonstrated by serial transplantation of marrow from engrafted recipients 11 months post transplant into new hosts for four additional months.

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.

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.

Long-term repopulating hematopoietic stem cells can be separated from cells which provided radioprotection (short-term repopulating cells) on the basis of size. This might be a result of the quiescent nature of long-term repopulating cells. To define the activity of these populations we utilized a dye, PKH26, which incorporates into the membrane of cells and is equally distributed to daughter cells when they divide. We were able to retrieve PKH26(+)-labeled cells posttransplant in the hematopoietic tissues of the recipients. We could also assess their cell cycle status and their ability, short- and long-term, to reconstitute secondary lethally irradiated hosts in limiting dilution. The results suggest that long-term repopulating cells remain quiescent in the bone marrow shortly after engraftment, whereas cells which radioprotect are more rapidly dividing. We could not detect labeled cells in the peripheral blood posttransplant, and even though cells homed to both the spleen and bone marrow the cells in the bone marrow were significantly more competent at reconstituting lethally irradiated secondary hosts.

We have previously demonstrated that we could separate long-term repopulating stem cells from cells that provided radioprotection (short-term repopulating cells) on the basis of size and suggested that this might be due to the quiescent nature of long-term repopulating cells. To further define the activity of these populations, we used a dye (PKH26), which incorporates into the membrane of cells and is equally distributed to daughter cells when they divide. We developed an assay, which allowed us to retrieve PKH26(+) long-term and short-term repopulating cells in the hematopoietic tissues of the recipients posttransplant. We were able to recover the labeled cells and determine their cell cycle activity, as well as their ability to reconstitute secondary lethally irradiated hosts in limiting dilution. The results of our assay suggest that long-term repopulating cells are quiescent in the bone marrow (BM) 48 hours after transplant. We were able to detect only a few labeled cells in the peripheral blood posttransplant and even though cells homed to both the spleen and BM, more long-term repopulating cells homed to the marrow and only these cells, which homed to the marrow, were capable of reconstituting lethally irradiated secondary hosts long-term.

T cell depletion (TCD) performed by elutriation has decreased the incidence of acute and chronic graft-versus-host disease (GvHD) following bone marrow transplantation (BMT). However, as with all forms of TCD, patients may experience graft failure (10%), delayed engraftment, and mixed chimerism. Because 66%-75% of the CD34+ cells coseparate with the small lymphocytes, which are removed by elutriation, we designed a phase I trial in HLA-identical siblings to determine if the readdition of these previously lost small CD34+ cells would improve elutriation's engraftment kinetics. CD34+ cells were isolated from the small cell fraction of 10 consecutive donor grafts and infused into the recipients along with the TCD graft. The positively selected product had a mean T cell content of 1.2 x 10(5)/kg and was 80% CD34+, doubling the CD34+ content of the graft. All patients engrafted promptly with a median time to 500 neutrophils/mm3, untransfused 50,000 platelets/mm3, and discharge from the hospital of 19 (range 10-25), 24 (14-52), and 24 (18-29) days, respectively. Acute GvHD occurred in 2 patients, and no patient had chronic GvHD. Augmenting stem cell dose may be an efficient and safe alternative for overcoming TCD-associated delayed engraftment and graft failure, rather than increasing immunosuppression.

Inactivation of the cyclin-dependent kinase inhibitors p16INK4A and p15INK4B are frequent alterations in neoplasia, often resulting from homozygous deletion or promoter region hypermethylation. We have analyzed both modes of inactivation of p15INK4B and p16INK4A in the major types of adult and pediatric hematological malignancies. Hypermethylation of p15INK4B, without alteration of p16INK4A, was an almost universal finding in adult acute myelogenous leukemia, and occurred very frequently in adult acute lymphocytic leukemia and pediatric acute myelogenous leukemia and acute lymphocytic leukemia. In contrast, neither p15INK4B nor p16INK4A were inactivated in any stage of chronic myelogenous leukemia. Hypermethylation of p16INK4A, often without alterations of p15INK4B, was found in non-Hodgkin's lymphoma and was much more frequent in cases with high-grade than low-grade histology. Enriched normal bone marrow stem cells had no detectable promoter region methylation of these genes, as analyzed by a newly developed PCR method. Remarkably distinct patterns of inactivation of p15INK4B and p16INK4A characterize different types of hematological malignancy, and alterations in these tumor suppressor genes are one of the most common alterations in hematological malignancies.

The purpose of this report is to demonstrate the phenotypic and functional characteristics of a primitive hematopoietic stem cell (HSC). We present evidence that an isolated murine HSC can repopulate the hematopoietic tissues of lethally irradiated recipient animals long term. By limiting dilution, as few as ten isolated stem cells can reconstitute mice for their lifetime. The stem cell which we have isolated does not copurify with colony forming unit-spleen or radioprotect recipients from lethal radiation. The phenotypic characterization of this rare cell, which represents 0.005% of total bone marrow, includes either the absence or very low expression of markers associated with long-term repopulating cells described by other groups. We believe this stem cell represents a very early self-renewing stem cell in the mouse.

The induction of apoptosis by the Fas/APO-1 receptor is important for T-cell-mediated cytotoxicity and down-regulation of immune responses. Binding of Fas ligand to the Fas/APO-1 receptor transduces an apoptotic signal that requires activation of interleukin 1beta-converting enzyme (ICE) and CPP32beta, members of a family of cysteine proteases that are evolutionarily conserved determinants of cell death. We report here that Fas/APO-1-triggered apoptosis involves ICE-mediated activation of p34cdc2 kinase. Ligation of the Fas receptor resulted in the rapid stimulation of ICE proteolytic activity and activation of p34cdc2 kinase. Specific tetrapeptide inhibitors of ICE (Acetyl-Tyr-Val-Ala-Asp-chloromethylketone) or CPP32beta (Acetyl-Asp-Glu-Val-Asp-aldehyde) prevented the anti-Fas antibody-mediated activation of p34cdc2 and inhibited apoptosis. Inhibition of p34cdc2 activity by transient overexpression of a dominant-negative cdc2 construct or human WEE1 kinase inhibited Fas-mediated apoptosis. These results suggest that activation of p34cdc2 kinase is a critical determinant of cell death mediated by Fas and ICE family proteases.

The induction of tumor cell death by anticancer therapy results from a genetic program of autonomous cell death termed apoptosis. Because the p53 tumor suppressor gene is a critical component for induction of apoptosis in response to DNA damage, its inactivation in cancers may be responsible for their resistance to genotoxic anticancer agents. The cellular response to DNA damage involves a cell-cycle arrest at both the G1/S and G2/M transitions; these checkpoints maintain viability by preventing the replication or segregation of damaged DNA. The arrest at the G1 checkpoint is mediated by p53-dependent induction of p21WAF1/CIP1, whereas the G2 arrest involves inactivation of p34cdc2 kinase. Following DNA damage, p53-deficient cells fail to arrest at G1 and accumulate at the G2/M transition. We demonstrate that abrogation of G2 arrest by caffeine-mediated activation of p34cdc2 kinase results in the selective sensitization of p53-deficient primary and tumor cells to irradiation-induced apoptosis. These data suggest that pharmacologic activation of p34cdc2 kinase may be a useful therapeutic strategy for circumventing the resistance of p53-deficient cancers to genotoxic anticancer agents.

The classical definition of lymphohematopoietic stem cells (LHSC), the most primitive progenitors of all blood cells, requires that they have the capacity for self-renewal and for the long-term production of all blood cell lineages. However, other characteristics of LHSC have been debated. Our previous data suggested that mouse LHSC are very slowly proliferating cells that generate delayed multilineage engraftment, while "radioprotection" (rapid engraftment that will prevent early death from radiation-induced marrow aplasia) results from more committed progenitors. Alternatively, some groups have reported that mouse LHSC are responsible for both radioprotection and long-term repopulation of all blood cell lineages. A possible explanation for this difference is that cells with the capacity for long-term production of all blood cell lineages are biologically heterogeneous. We now show that 10 LHSC can generate all blood cell lineages for the lifetime of the animal. However, these cells lacked radioprotection and spleen colony-forming activity. LHSC were identified and isolated by their small size, their lack of expression of antigens characteristic of mature blood cell lineages, and their high expression of aldehyde dehydrogenase. In addition, these cells were found to express undetectable or low levels of many antigens presumed to mark LHSC, including Thy-1, Ly-6A/E (Sca-1), c-kit, and CD34. There appears to be at least two classes of LHSC with the capacity for long-term production of all blood cell lineages: one that generates both radioprotection and long-term engraftment and one that produces delayed but durable engraftment. Our data suggest that this latter class may represent a very primitive class of LHSC.

Estrogen appears to be a negative regulator of normal hematopoiesis. Chromosome 6q, which contains the estrogen receptor (ER) gene, is frequently altered in human hematopoietic neoplasms. The ER gene, which has growth and metastasis suppressor activity in many different cell types, is inactivated by promoter methylation in some ER-negative breast tumors and 100% of colorectal tumors. We now report that the promoter region of the ER gene is aberrantly methylated in 86% of human hematopoietic tumors, including 8 of 9 pediatric acute lymphocytic leukemia, 17 of 18 adult acute lymphocytic leukemia, 21 of 23 adult acute myelogenous leukemia, 3 of 6 chronic phase chronic myelogenous leukemia, 9 of 9 blast crisis chronic myelogenous leukemia and 5 of 8 lymphomas. This methylation event was also present in all nine leukemia cell lines examined, where it was associated with very low or absent ER expression. In addition, rat and mouse leukemia cell line also exhibited this change, indicating that ER CpG island methylation in leukemias is conserved among species. Our results suggest that ER CpG island methylation could be an important step in the genesis of human hematopoietic neoplasms and might be a useful molecular marker for monitoring the clinical status of these diseases.

Expression of the BCR-ABL chimeric gene in chronic myeloid leukemia results in the inhibition of apoptosis, a genetically programmed process of autonomous cell death. BCR-ABL and other genetic factors that suppress apoptosis confer cross-resistance to cytotoxic agents with diverse mechanisms of action. Eradication of the chronic myeloid leukemia clone requires strategies that circumvent this inherent resistance to cytotoxic therapy. We have determined that BCR-ABL expression augments the sensitivity of hematopoietic cells to growth factor-mediated signals of differentiation; hematopoietic growth factors induce the selective terminal differentiation of chronic myeloid leukemia progenitors at concentrations that allow optimal growth of normal progenitors. Hematopoietic growth factors may be an effective strategy for the elimination of cytotoxic therapy-resistant leukemic cells by inducing their terminal differentiation while allowing concomitant expansion of coexistent normal hematopoietic progenitors.

CD34 is expressed on human hematopoietic stem and progenitor cells, and its clinical usefulness for the purification of stem cells has been well established. However, a similar pattern of expression for murine CD34 (mCD34) has not yet been determined. Two polyclonal anti-mCD34 antibodies that specifically recognize both endogenous and recombinant murine CD34 were developed to characterize the mCD34 protein and to determine its pattern of expression on murine cell lines and hematopoietic progenitor cells. Fluorescence-activated cell sorter analysis showed that mCD34 is expressed on NIH/3T3 embryonic fibroblasts, PA6 stromal cells, embryonic stem cells, M1 leukemia cells, and a subpopulation of normal bone marrow cells. Murine CD34 was found to be a glycoprotein expressed on the cell surface as either a full-length (approximately 100 kD) or truncated (approximately 90 kD) protein in NIH/3T3 and PA6 cells. Recombinant full-length CD34, when expressed in the CHO-K1 cell line, had a molecular weight of approximately 105 kD. Full-length CD34 expressed on M1 leukemia cells, had a higher apparent molecular weight (110 kD). These results suggest that there are glycosylation differences between CD34 expressed by different cell types. The full-length form, but not the truncated form, is a phosphoprotein that is hyperphosphorylated in response to 12-0-Tetradecanoyl phorbol 13-acetate treatment, suggesting potential functional differences between the two forms. Selection of the 3% highest-expressing CD34+ bone marrow cells enriched for the hematopoietic precursors that form colony-forming unit-spleen (CFU-S), CFU-granulocyte-macrophage, and burst-forming unit-erythroid. Transplantation of lethally irradiated mice with these cells demonstrated both short- and long-term repopulating ability, indicating that this population contains both functional hematopoietic progenitors and the putative stem cell. These antibodies should be useful to select for murine hematopoietic stem cells.

BCR-ABL expression is presumed to effect clonal expansion in chronic myeloid leukemia (CML) by deregulation of cell proliferation. However, most studies have found that relative rates of cell proliferation are not increased in CML. Moreover, we found that CML progenitors display a normal proliferative response to growth factors and do not manifest greater proliferative potential than normal progenitors. Growth of malignancies depends on an imbalance between the rate of cell production and the rate of cell death. We found that BCR-ABL expression inappropriately prolongs the growth factor-independent survival of CML myeloid progenitors and granulocytes by inhibiting apoptosis, a genetically programmed process of active cell death; inhibition of BCR-ABL expression by antisense oligonucleotides reversed the suppression of apoptosis as well as the enhancement of survival. The decreased rate of programmed cell death appears to be the primary mechanism by which BCR-ABL effects expansion of the leukemic clone in CML.

Chronic myeloid leukemia (CML) is characterized by an initial chronic phase of expanded yet orderly clonal hematopoiesis that is distinguished by the BCR-ABL gene rearrangement. We found that although the mature myeloid compartment in patients with CML was expanded and entirely derived from the dominant leukemic clone, the primitive hematopoietic progenitor compartment did not show a corresponding expansion and was substantially enriched for cells without the BCR-ABL gene rearrangement. More importantly, primitive progenitors exhibiting the BCR-ABL gene rearrangement did not express either the BCR-ABL hybrid mRNA or fusion protein (P210). Expression of P210 protein and BCR-ABL mRNA increased with myeloid commitment in vivo as well as with growth factor-induced proliferation and differentiation of the primitive CML progenitors in vitro. This differential expression of BCR-ABL between primitive and mature CML progenitors may explain the expansion of the leukemic clone at the level of mature myeloid progenitors and granulocytes without a concomitant expansion of primitive CML progenitors. Because BCR-ABL mRNA is minimally expressed or may be absent in primitive CML progenitors, these cells may escape detection by reverse transcriptase-polymerase chain reaction and eradication by antisense oligonucleotides targeted against BCR-ABL mRNA.

The mouse Y-specific DNA sequence pY2 was used as a probe for fluorescence in situ hybridization (FISH) to evaluate murine hematopoietic tissues after sex-mismatched bone marrow transplant (BMT). The pY2 probe was localized to the long arm of the Y chromosome on BM metaphases. Hybridization of pY2 in FISH of interphase cells from BM, spleen, and thymus after BMT was compared with Southern blot analysis; both methods gave comparable results. Only FISH was able to analyze post-BMT peripheral blood (PB) samples successfully, and provides a useful method for following engraftment status in the mouse on an ongoing basis.

Long-term reconstitution of the lymphohaematopoietic cells of a mouse after lethal irradiation requires the transplantation of at least (5-10) x 10(3) bone marrow cells. Several cell-separation techniques based on cell-surface characteristics have been used in attempts to identify the pluripotent haematopoietic stem cells (PHSC), and have allowed the long-term engraftment of lethally irradiated mice with an enriched fraction of fewer than 200 marrow cells. But these techniques enrich not only for PHSC but also for haematopoietic progenitors, especially day-12 spleen colony-forming units (CFU-S). Although day-12 CFU-S have been postulated to be primitive multipotential haematopoietic progenitors, with day-8 CFU-S representing later, more committed progenitors, recent evidence suggests that neither of these CFU-S represents mouse PHSC. Here we report that counterflow centrifugal elutriation, which sorts cells on the basis of size and density, can separate PHSC from these less primitive progenitors. The fraction containing the largest cells was enriched for the granulocyte-macrophage colony-forming units (CFU-GM), but gave only transient, early engraftment and was therefore depleted of PHSC. The intermediate fraction was enriched for CFU-S, but depleted of CFU-GM. Despite being devoid of CFU-GM and CFU-S, the fraction consisting of only morphological lymphocytes gave sustained, albeit delayed, reconstitution of all lymphohaematopoietic cells, and was therefore enriched for PHSC. We conclude that there are two vital classes of engrafting cells: committed progenitors, which provide initial, unsustained engraftment, and PHSC, which produce delayed, but durable, engraftment. Therefore for late haematological reconstitution, PHSC must be transplanted with a distinguishable source of early engrafting cells, thereby allowing the lethally irradiated host to survive initial aplasia.

Since enrichment of human bone-marrow hematopoietic progenitors is becoming more feasible and since purified growth factors are now available, we sought to study the action of growth factors on CD34-positive enriched cultures of human bone-marrow cells. We tested the effect of recombinant human (rh) granulocyte-macrophage colony-stimulating factor (GM-CSF), rh interleukin-3 (IL-3), or a unique biologic response modifier, bryostatin 1, on the growth of purified CD34 cells obtained by limiting dilution in single-cell cultures. We have shown previously that bryostatin 1 stimulates both myeloid and erythroid progenitors of human origin in vitro. In this study both IL-3 and GM-CSF supported colony formation from 500, 100, or single-cell cultures at equivalent plating efficiences, suggesting a direct action of these factors on hematopoietic cell growth. Conversely, bryostatin 1 did not support the growth of CD34 cells in single-cell cultures, and the cloning efficiency increased with increasing the number of cells in the culture. To test whether the indirect action of bryostatin 1 might be mediated through the production of growth factors by accessory cells, studies were performed using antibodies directed against human IL-3 and GM-CSF in culture with bryostatin 1 and normal human bone-marrow cells. Results are consistent with the hypothesis that bryostatin 1 could have a stimulatory effect on the accessory cell populations to produce either IL-3 or GM-CSF. Further support for this notion was obtained by demonstrating that T cells, which are cells known to be able to produce IL-3 and GM-CSF, are stimulated by bryostatin 1 to express messenger RNA (mRNA) for specific growth factors, including GM-CSF. These results provide further support that bryostatin 1 may be a useful clinical agent to stimulate hematopoiesis in vivo.

Bryostatin 1, a macrocyclic lactone isolated from the marine bryozoan Bugula neritina, has demonstrated both antineoplastic activity against the murine P388 leukemia line in vivo and stimulatory activity against mouse and human hematopoietic progenitors. We studied the effects of bryostatin 1 on the growth of human leukemias in vitro. Bryostatin 1 inhibited 1 to 4 logs of clonogenic leukemia cell growth from three of four leukemia cell lines. Bryostatin 1 also inhibited, by at least 1 log, the proliferation of clonogenic acute nonlymphocytic leukemia (ANLL) cells from 10 to 12 patients with newly diagnosed or relapsed ANLL. Maximal inhibition of leukemic growth occurred at 10(-9) to 10(-7) mol/L bryostatin 1. Interestingly, bryostatin 1 also inhibited the growth of hematopoietic progenitors from eight patients with myelodysplastic syndromes (MDS). Leukemia cells exposed to bryostatin 1 for up to 96 hours and then washed, demonstrated no substantial inhibition of clonogenic growth, indicating that the anti-leukemic effect of bryostatin 1 is cytostatic. The phorbol ester 12-0-tetradecanoylphorbol-13-acetate (TPA) produced more potent inhibition of clonogenic leukemia growth, and this inhibition was blocked by bryostatin 1. Thus, the anti-leukemic activity of bryostatin 1 may be mediated through activation of protein kinase C. Bryostatin 1 inhibits clonogenic leukemia cells at concentrations that stimulate normal hematopoietic progenitors. The differential effects of bryostatin 1 on normal and abnormal hematopoiesis suggest that bryostatin 1 may have value in the treatment of leukemias and MDS.

Defective stem cells of WBB6F1-W/Wv mice produce macrocytic red blood cells (RBCs); stem cells of WBB6F1-+/+ mice produce normocytic RBCs. Utilization of the Coulter counter channelyzer permitted good dissociation between the size distribution of populations of +/+ and W/Wv RBCs. Peaks (mean cell volumes) for +/+ and W/Wv RBCs have been determined to be between the 30th and 40th channel and 50th and 60th channel, respectively. Variability of profiles for individual mice of both genotypes did not exceed the variability of separate determinations of the same cell suspension from a single mouse. Admixture (approximately 15%) of either type of erythrocytes could be quantitatively detected by this method. One week after transplant of 10(7) +/+ marrow cells into W/Wv recipients, 25% of donor type erythrocytes were detected. Eighteen days post-graft, concentration of +/- normocytes exceeded the concentration of macrocytes in the W/Wv recipients' circulation. Approximately 45 days post-transplant, the proportion of macrocytes decreased below the 10% detectable level. Calculation of the daily RBC production rate during repopulation and estimation of the number of RBCs produced by a single hematopoietic colony were determined. The RBC size profile was found to be a convenient method for studying the effect of implantation of W/Wv marrow into lethally irradiated +/+ mice. This method proved suitable for repetitive determination of the size population in individual transplanted mice.