Reprint of: Preclinical characterization of DUOC-01, a cell therapy product derived from banked umbilical cord blood for use as an adjuvant to umbilical cord blood transplantation for treatment of inherited metabolic diseases.

BACKGROUND AIMS: Cord blood (CB) transplantation slows neurodegeneration during certain inherited metabolic diseases. However, the number of donor cells in the brain of patients does not appear to be sufficient to provide benefit until several months after transplant. We developed the cell product DUOC-01 to provide therapeutic effects in the early post-transplant period. METHODS: DUOC-01 cultures initiated from banked CB units were characterized by use of time-lapse photomicroscopy during the 21-day manufacturing process. Antigen expression was measured by means of flow cytometry and immunocytochemistry; transcripts for cytokines and enzymes by quantitative real-time polymerase chain reaction; activities of lysosomal enzymes by direct biochemical analysis; alloreactivity of DUOC-01 and of peripheral blood (PB) mononuclear cells (MNC) to DUOC-01 by mixed lymphocyte culture methods; and cytokine secretion by Bioplex assays. RESULTS: DUOC-01 cultures contained highly active, attached, motile, slowly proliferating cells that expressed common (cluster of differentiation [CD]11b, CD14 and Iba1), M1 type (CD16, inducible nitric oxide synthase), and M2-type (CD163, CD206) macrophage or microglia markers. Activities of 11 disease-relevant lysosomal enzymes in DUOC-01 products were similar to those of normal PB cells. All DUOC-01 products secreted interleukin (IL)-6 and IL-10. Accumulation of transforming growth factor-ß, IL-1ß, interferon-γ and TNF-α in supernatants was variable. IL-12, IL-2, IL-4, IL-5 and IL-13 were not detected at significant concentrations. Galactocerebrosidase, transforming growth factor-ß and IL-10 transcripts were specifically enriched in DUOC-01 relative to CB cells. PB MNCs proliferated and released cytokines in response to DUOC-01. DUOC-01 did not proliferate in response to mismatched MNC. CONCLUSIONS: DUOC-01 has potential as an adjunctive cell therapy to myeloablative CB transplant for treatment of inherited metabolic diseases.

Preclinical characterization of DUOC-01, a cell therapy product derived from banked umbilical cord blood for use as an adjuvant to umbilical cord blood transplantation for treatment of inherited metabolic diseases.

BACKGROUND AIMS: Cord blood (CB) transplantation slows neurodegeneration during certain inherited metabolic diseases. However, the number of donor cells in the brain of patients does not appear to be sufficient to provide benefit until several months after transplant. We developed the cell product DUOC-01 to provide therapeutic effects in the early post-transplant period. METHODS: DUOC-01 cultures initiated from banked CB units were characterized by use of time-lapse photomicroscopy during the 21-day manufacturing process. Antigen expression was measured by means of flow cytometry and immunocytochemistry; transcripts for cytokines and enzymes by quantitative real-time polymerase chain reaction; activities of lysosomal enzymes by direct biochemical analysis; alloreactivity of DUOC-01 and of peripheral blood (PB) mononuclear cells (MNC) to DUOC-01 by mixed lymphocyte culture methods; and cytokine secretion by Bioplex assays. RESULTS: DUOC-01 cultures contained highly active, attached, motile, slowly proliferating cells that expressed common (cluster of differentiation [CD]11b, CD14 and Iba1), M1 type (CD16, inducible nitric oxide synthase), and M2-type (CD163, CD206) macrophage or microglia markers. Activities of 11 disease-relevant lysosomal enzymes in DUOC-01 products were similar to those of normal PB cells. All DUOC-01 products secreted interleukin (IL)-6 and IL-10. Accumulation of transforming growth factor-ß, IL-1ß, interferon-γ and TNF-α in supernatants was variable. IL-12, IL-2, IL-4, IL-5 and IL-13 were not detected at significant concentrations. Galactocerebrosidase, transforming growth factor-ß and IL-10 transcripts were specifically enriched in DUOC-01 relative to CB cells. PB MNCs proliferated and released cytokines in response to DUOC-01. DUOC-01 did not proliferate in response to mismatched MNC. CONCLUSIONS: DUOC-01 has potential as an adjunctive cell therapy to myeloablative CB transplant for treatment of inherited metabolic diseases.

Synthesis and preliminary evaluation of n.c.a. iodoquine: a novel radiotracer with high uptake in cells with high ALDH1 expression.

PURPOSE: Chloroquine has demonstrated high affinity for aldehyde dehydrogenase 1A1 (ALDH1), an enzyme expressed in the highly tumorigenic CD133+ brain tumor initiating subpopulation. The purpose of this study is to report the novel synthesis of a chloroquine analogue, n.c.a. iodoquine, and the in vitro and in vivo uptake in cells with high ALDH1 content. METHODS AND MATERIALS: Iodoquine was synthesized in novel no-carrier-added forms (n.c.a.) for both 125I and 123I. I25I IQ and 18F FDG cell uptake assays were performed in the L1210 and L1210cpa (cyclophosphamide resistant), A549, and MG456 glioblastoma cell lines. Uptake was expressed as a percent of the administered activity. 125I IQ biodistribution studies assessed organ uptake at 1, 4, and 24 hours after IV administration (n= 15 total; 5 mice/timepoint). Radiation dosimetry estimates were calculated using standard OLINDA/EXM software. In vivo imaging of 123I IQ uptake in MG456 glioblastoma mouse model (n=10) was performed with small animal high resolution micro-SPECT. Autoradiography and histology co-localized radiotracer and tumor biodistribution. Uptake in MG456 glioblastoma tumors was quantified with gamma counting. RESULTS: L1210 cpa (high ALDH1) showed significantly higher 125I IQ uptake compared to the parental L1210 (low ALDH1) for all time points through 4 hours (20.7% ± 1.4% versus 11.0% ± 0.5%; 21.3% ± 0.9% versus 11.0% ± 0.4%; 20.6% ± 0.7% versus 9.4% ± 0.3%; and 15.7% ± 0.7% versus 7.5% + 0.4% at 30 minutes, and 1, 2 and 4 hours, respectively; p < 0.001 for all time points). In the CD133+ fraction of MG456 glioblastoma cell line, IQ uptake was significantly higher compared to FDG at all time points through 4 hours (81.5% ± 0.9% versus 1.3% ± 0.1%; 88.8% ± 0.4% versus 1.3% ± 0.1%; 87.8% ± 2.1% versus 1.7% ± 0.2%; and 87.0% ± 2.4% versus 1.8% ± 0.1 at 30 minutes, and 1, 2 and 4 hours, respectively; p > 0.001 for all time points). The A549 lung cancer cell line also showed high IQ uptake through 4 hours. IQ normal biodistribution studies showed rapid renal excretion and very low normal background brain activity after IV administration. In vivo micro-SPECT images showed mild uptake in larger MG456 glioblastomas (n=6) as verified with autoradiography and histology. Gamma well counter uptake in large tumors was 2.3% ± 0.48% ID/g (n=5). CONCLUSION: Iodoquine localizes to cells with high ALDH1 content. Cell assays show high 125I IQ uptake in the MG456 cell line, and in vivo micro-SPECT imaging showed mild 123I IQ uptake in MG456 glioblastomas. Further studies are necessary to investigate 131I IQ as a potential therapeutic agent targeting the highly tumorigenic CD133+ brain tumor stem cell subpopulation.

Increased numbers of total nucleated and CD34+ cells in blood group O cord blood: an analysis of neonatal innate factors in the Korean population.

BACKGROUND: We analyzed neonatal factors that could affect hematopoietic variables of cord blood (CB) donated from Korean neonates. STUDY DESIGN AND METHODS: The numbers of total nucleated cells (TNCs), CD34+ cells, and CD34+ cells/TNCs of CB in neonates were compared according to sex, gestational age, birth weight, birth weight centile for gestational age, and ABO blood group. RESULTS: With 11,098 CB units analyzed, blood group O CB showed an increased number of TNCs, CD34+ cells, and CD34+ cells/TNCs compared with other blood groups. Although TNC counts were lower in males, no difference in the number of CD34+ cells was demonstrated because the number of CD34+ cells/TNCs was higher in males. An increase in the gestational age resulted in an increase in the number of TNCs and decreases in the number of CD34+ cells and CD34+ cells/TNCs. The numbers of TNCs, CD34+ cells, and CD34+ cells/TNCs increased according to increased birth weight centile as well as birth weight. CONCLUSION: CB with blood group O has unique hematologic variables in this large-scale analysis of Korean neonates, although the impact on the storage policies of CB banks or the clinical outcome of transplantation remains to be determined.

Feasibility of low-dose interleukin-2 therapy following T-cell-depleted nonmyeloablative allogeneic hematopoietic stem cell transplantation from HLA-matched or -mismatched family member donors.

INTRODUCTION: High relapse rates and infections remain primary causes of failure in nonmyeloablative transplantation. Interleukin-2 (IL-2) may stimulate the immune system and improve outcomes. The primary objective of this pilot study was to evaluate the feasibility of administering IL-2 following a T-cell-depleted nonmyeloablative hematopoietic stem cell transplant. METHODS: Patients received T-cell-depleted nonmyeloablative transplant from a matched or mismatched related donor. Those with allogeneic engraftment,

Progenitor cell dose determines the pace and completeness of engraftment in a xenograft model for cord blood transplantation.

Two critical concerns in clinical cord blood transplantation are the initial time to engraftment and the subsequent restoration of immune function. These studies measured the impact of progenitor cell dose on both the pace and strength of hematopoietic reconstitution by transplanting nonobese diabetic/severe combined immunodeficiency/interleukin-2 receptor-gamma-null (NSγ) mice with lineage-depleted aldehyde dehydrogenase-bright CD34(+) human cord blood progenitors. The progress of each transplant was monitored over an extended time course by repeatedly analyzing the peripheral blood for human hematopoietic cells. In vivo human hematopoietic development was complete. After long-term transplantation assays (≥ 19 weeks), human T-cell development was documented within multiple tissues in 16 of 32 NSγ mice. Human T-cell differentiation was active within NSγ thymuses, as documented by the presence of CD4(+) CD8(+) T-cell progenitors as well as T-cell receptor excision circles. It is important to note that although myeloid and B-cell engraftment was detected as early as 4 weeks after transplantation, human T-cell development was exclusively late onset. High progenitor cell doses were associated with a robust human hematopoietic chimerism that accelerated both initial time to engraftment and subsequent T-cell development. At lower progenitor cell doses, the chimerism was weak and the human hematopoietic lineage development was frequently incomplete.

Human umbilical cord blood endothelial progenitor cells decrease vein graft neointimal hyperplasia in SCID mice.

AIMS: Vein graft endothelial damage is a key step in the development of neointimal hyperplasia, leading to vein graft failure. We sought to determine whether exogenous endothelial progenitor cells could promote vein graft re-endothelialization, and thereby ameliorate neointimal hyperplasia. METHODS AND RESULTS: Carotid artery interposition grafting was performed with syngeneic inferior vena cavae in mice with severe combined immunodeficiency (SCID). Lineage-negative human umbilical cord blood (hUCB) cells (or medium alone) were injected into vein-grafted mice intra-operatively and 2 weeks post-operatively. In vein grafts from hUCB cell-injected mice, we found human HLA-expressing endothelial cells, as well as increased levels of VEGF and FGF-2. Furthermore, hUCB cells secreted VEGF and FGF-2 in vitro. The markedly enhanced endothelial regeneration, likely resulting from both direct engraftment and paracrine actions of hUCB cells, inhibited inflammatory response, diminished intimal cell proliferation, and reduced neointimal hyperplasia in the vein grafts. CONCLUSIONS: hUCB cells may accelerate vein graft re-endothelialization via both direct differentiation into endothelial cells and release of paracrine factors to enhance endothelial regeneration and reduce inflammation. These data highlight a potential therapeutic role for cellular therapy in vessel injury.

Natural killer cell-enriched donor lymphocyte infusions from A 3-6/6 HLA matched family member following nonmyeloablative allogeneic stem cell transplantation.

Infusing natural killer (NK) cells following transplantation may allow less infections and relapse with little risk of acute graft-versus-host disease (aGVHD). We delivered 51 total NK cell-enriched donor lymphocyte infusions (DLIs) to 30 patients following a 3-6/6 HLA matched T cell-depleted nonmyeloablative allogeneic transplant. The primary endpoint of this study was feasibility and safety. Eight weeks following transplantation, donor NK cell-enriched DLIs were processed using a CD56(+) selecting column with up to 3 fresh infusions allowed. Toxicity, relapse, and survival were monitored. T cell phenotype, NK cell functional recovery, and KIR typing were assessed for association with outcomes. Fourteen matched and 16 mismatched transplanted patients received a total of 51 NK cell-enriched DLIs. Selection resulted in 96% (standard deviation [SD] 8%) purity and 83% (SD 21%) yield in the matched setting and 97% (SD 3%) purity and 77% (SD 24%) yield in the mismatched setting. The median number of CD3(-) CD56(+) NK cells infused was 10.6 (SD 7.91) x 10(6) cells/kg and 9.21 (SD 5.6) x 10(6) cells/kg, respectively. The median number of contaminating CD3(+)CD56(-) T cells infused was .53 (1.1) x 10(6) and .27 (.78) x 10(6) in the matched and mismatched setting, respectively. Only 1 patient each in the matched (n = 14) or mismatched (n = 16) setting experienced severe aGVHD with little other toxicity attributable to the infusions. Long-term responders with multiple NK cell-enriched infusions and improved T cell phenotypic recovery had improved duration of responses (p = .0045) and overall survival (OS) (P = .0058). A 1-step, high-yield process is feasible, and results in high doses of NK cells infused with little toxicity. NK cell-enriched DLIs result in improved immune recovery and outcomes for some. Future studies must assess whether the improved outcomes are the direct result of the high doses and improved NK cell function or other aspects of immune recovery.

Inhibition of aldehyde dehydrogenase expands hematopoietic stem cells with radioprotective capacity.

Hematopoietic stem cells (HSCs) are enriched for aldehyde dehydrogenase (ALDH) activity and ALDH is a selectable marker for human HSCs. However, the function of ALDH in HSC biology is not well understood. We sought to determine the function of ALDH in regulating HSC fate. Pharmacologic inhibition of ALDH with diethylaminobenzaldehyde (DEAB) impeded the differentiation of murine CD34(-)c-kit(+)Sca-1(+)lineage(-) (34(-)KSL) HSCs in culture and facilitated a ninefold expansion of cells capable of radioprotecting lethally irradiated mice compared to input 34(-)KSL cells. Treatment of bone marrow (BM) 34(-)KSL cells with DEAB caused a fourfold increase in 4-week competitive repopulating units, verifying the amplification of short-term HSCs (ST-HSCs) in response to ALDH inhibition. Targeted siRNA of ALDH1a1 in BM HSCs caused a comparable expansion of radioprotective progenitor cells in culture compared to DEAB treatment, confirming that ALDH1a1 was the target of DEAB inhibition. The addition of all trans retinoic acid blocked DEAB-mediated expansion of ST-HSCs in culture, suggesting that ALDH1a1 regulates HSC differentiation via augmentation of retinoid signaling. Pharmacologic inhibition of ALDH has therapeutic potential as a means to amplify ST-HSCs for transplantation purposes.

Targeting aldehyde dehydrogenase: a potential approach for cell labeling.

INTRODUCTION: To advance the science and clinical application of stem cell therapy, the availability of a highly sensitive, quantitative and translational method for tracking stem cells would be invaluable. Because hematopoetic stem cells express high levels of the cytosolic enzyme aldehyde dehydrogenase-1A1 (ALDH1), we sought to develop an agent that is specific to ALDH1 and thus to cells expressing the enzyme. Such an agent might be also helpful in identifying tumors that are resistant to cyclophosphomide chemotherapy because ALDH1 is known to be responsible for this resistance. METHODS: We developed schemes for the synthesis of two radioiodinated aldehdyes - N-formylmethyl-5-[*I]iodopyridine-3-carboxamide ([*I]FMIC) and 4-diethylamino-3-[*I]iodobenzaldehyde ([*I]DEIBA)-at no-carrier-added levels from their respective tin precursors. These agents were evaluated using pure ALDH1 and tumor cells that expressed the enzyme. RESULTS: The average radiochemical yields for the synthesis of [(125)I]FMIC and [(125)I]DEIBA were 70+/-5% and 47+/-14%, respectively. ALDH1 converted both compounds to respective acids suggesting their suitability as ALDH1 imaging agents. Although ability of ALDH1 within the cells to oxidize one of these substrates was shown, specific uptake in ALDH-expressing tumor cells could not be demonstrated. CONCLUSION: To pursue this approach for ALDH1 imaging, radiolabeled aldehydes need to be designed such that, in addition to being good substrates for ALDH1, the cognate products should be sufficiently polar so as to be retained within the cells.

A role for E2F activities in determining the fate of Myc-induced lymphomagenesis.

The phenotypic heterogeneity that characterizes human cancers reflects the enormous genetic complexity of the oncogenic process. This complexity can also be seen in mouse models where it is frequently observed that in addition to the initiating genetic alteration, the resulting tumor harbors additional, somatically acquired mutations that affect the tumor phenotype. To investigate the role of genetic interactions in the development of tumors, we have made use of the Emu-myc model of pre-B and B cell lymphoma. Since various studies point to a functional interaction between Myc and the Rb/E2F pathway, we have investigated the role of E2F activities in the process of Myc-induced lymphomagenesis. Whereas the absence of E2F1 and E2F3 function has no impact on Myc-mediated tumor development, the absence of E2F2 substantially accelerates the time of tumor onset. Conversely, tumor development is delayed by the absence of E2F4. The enhanced early onset of tumors seen in the absence of E2F2 coincides with an expansion of immature B lineage cells that are likely to be the target for Myc oncogenesis. In contrast, the absence of E2F4 mutes the response of the lineage to Myc and there is no expansion of immature B lineage cells. We also find that distinct types of tumors emerge from the Emu-myc mice, distinguished by different patterns of gene expression, and that the relative proportions of these tumor types are affected by the absence of either E2F2 or E2F4. From these results, we conclude that there are several populations of tumors that arise from the Emu-myc model, reflecting distinct populations of cells that are susceptible to Myc-mediated oncogenesis and that the proportion of these cell populations is affected by the presence or absence of E2F activities.

Cytokine profile of human adipose-derived stem cells: expression of angiogenic, hematopoietic, and pro-inflammatory factors.

Adipose tissue serves as a source of adipokines and cytokines with both local and systemic actions in health and disease. In this study, we examine the hypothesis that multipotent human adipose-derived stem cells (ASCs), capable of differentiating along the adipocyte, chondrocyte, and osteoblast pathways, contribute to adipose tissue-derived cytokine secretion. Following exposure to basic fibroblast growth factor (bFGF) or epidermal growth factor (EGF), the ASCs significantly increase their secretion of hepatocyte growth factor (HGF), a cytokine implicated in hematopoiesis, vasculogenesis, and mammary epithelial duct formation. Ascorbic acid synergizes with these inductive factors, further increasing HGF levels. Following exposure to lipopolysaccharide, ASCs increase their secretion of both hematopoietic (granulocyte/monocyte, granulocyte, and macrophage colony stimulating factors, interleukin 7) and proinflammatory (interleukins 6, 8, and 11, tumor necrosis factor alpha) cytokines based on ELISA and RT-PCR. In co-cultures established with umbilical cord blood-derived CD34(+) cells, the ASCs support long-term hematopoiesis in vitro. Furthermore, in short-term 12-day co-cultures, the ASC maintain and expand the numbers of both myeloid and lymphoid progenitors. These observations are consistent with the functionality of the secreted cytokines and confirm recent reports by other laboratories concerning the hematopoietic supportive capability of ASCs. We conclude that the ASCs display cytokine secretory properties similar to those reported for bone marrow-derived mesenchymal stem cells (MSCs).

Extended passaging, but not aldehyde dehydrogenase activity, increases the chondrogenic potential of human adipose-derived adult stem cells.

Adipose-derived adult stem (ADAS) cells represent an abundant population of multipotent mesodermal cells residing in various adipose tissue depots. ADAS cell preparations appear heterogeneous, yet at a clonal level, greater than 50% of these cells exhibit multilineage differentiation potential. To date, there have been few attempts to define prospectively a homogenous population of multipotent cells. In this study, we investigated whether aldehyde dehydrogenase (ALDH) can be used to enrich ADAS cells with increased chondrogenic potential. ALDH has been previously used to isolate primitive hematopoietic progenitors and has been implicated in early neurogenesis. Human ADAS cells were purified based on ALDH activity, and the cells were expanded and induced for chondrogenic differentiation using BMP-6 in a 3-D alginate culture. No significant differences in chondrogenic potential were observed in the ALDH-positive cells compared to unsorted controls. In contrast, significant differences were noted between cells assayed at passage 4 (P4) and cells assayed at passage 9 (P9). Following BMP-6 induction, AGC1 gene expression in P9 cells increased 290-fold over P4 cells. Similarly, COL2A1 expression in P9 cells increased fivefold compared to P4 cells, while COL10A1 levels remained unchanged. Immunohistochemical analysis over 28 days revealed consistent findings at the protein level for collagen II, collagen X, and aggrecan. No changes in telomerase activity were detected across passage, suggesting that ADAS cells retain some level of "stemness" in monolayer culture. These findings suggest that the chondrogenic potential of ADAS cells increases with passage number, although ALDH may not be a suitable marker for chondrogenesis.

The immunogenicity of human adipose-derived cells: temporal changes in vitro.

Regenerative medical techniques will require an abundant source of human adult stem cells that can be readily available at the point of care. The ability to use unmatched allogeneic stem cells will help achieve this goal. Since adipose tissue represents an untapped reservoir of human cells, we have compared the immunogenic properties of freshly isolated, collagenase-digested human adipose tissue-derived stromal vascular fraction cells (SVFs) relative to passaged, plastic-adherent adipose-derived stem cells (ASCs). Parallel studies have shown that adherence to plastic and subsequent expansion of human adipose-derived cells selects for a relatively homogeneous cell population based on immunophenotype. Consistent with these findings, the presence of hematopoietic-associated markers (CD11a, CD14, CD45, CD86, and histocompatible locus antigen-DR [HLA-DR]) detected on the heterogeneous SVF cell population decreased upon subsequent passage of the ASCs. In mixed lymphocyte reactions (MLRs), SVFs, and early passage ASCs stimulated proliferation by allogeneic responder T cells. In contrast, the ASCs beyond passage P1 failed to elicit a response from T cells. Indeed, late passage ASCs actually suppressed the MLR response. Although these results support the feasibility of allogeneic human ASC transplantation, confirmatory in vivo animal studies will be required.

Immunophenotype of human adipose-derived cells: temporal changes in stromal-associated and stem cell-associated markers.

Adipose tissue represents an abundant and accessible source of multipotent adult stem cells and is used by many investigators for tissue engineering applications; however, not all laboratories use cells at equivalent stages of isolation and passage. We have compared the immunophenotype of freshly isolated human adipose tissue-derived stromal vascular fraction (SVF) cells relative to serial-passaged adipose-derived stem cells (ASCs). The initial SVF cells contained colony-forming unit fibroblasts at a frequency of 1:32. Colony-forming unit adipocytes and osteoblasts were present in the SVF cells at comparable frequencies (1:28 and 1:16, respectively). The immunophenotype of the adipose-derived cells based on flow cytometry changed progressively with adherence and passage. Stromal cell-associated markers (CD13, CD29, CD44, CD63, CD73, CD90, CD166) were initially low on SVF cells and increased significantly with successive passages. The stem cell-associated marker CD34 was at peak levels in the SVF cells and/or early-passage ASCs and remained present, although at reduced levels, throughout the culture period. Aldehyde dehydrogenase and the multidrug-resistance transport protein (ABCG2), both of which have been used to identify and characterize hematopoietic stem cells, are expressed by SVF cells and ASCs at detectable levels. Endothelial cell-associated markers (CD31, CD144 or VE-cadherin, vascular endothelial growth factor receptor 2, von Willebrand factor) were expressed on SVF cells and did not change significantly with serial passage. Thus, the adherence to plastic and subsequent expansion of human adipose-derived cells in fetal bovine serum-supplemented medium selects for a relatively homogeneous cell population, enriching for cells expressing a stromal immunophenotype, compared with the heterogeneity of the crude SVF.

Distinct hematopoietic progenitor compartments are delineated by the expression of aldehyde dehydrogenase and CD34.

A broad range of hematopoietic stem cells and progenitors reside within a fraction of umbilical cord blood (UCB) that exhibits low light scatter properties (SSC(lo)) and high expression of aldehyde dehydrogenase (ALDH(br)). Many SSC(lo) ALDH(br) cells coexpress CD34; however, other cells express either ALDH or CD34. To investigate the developmental potential of these cell subsets, purified ALDH(br) CD34+, ALDH(neg) CD34+, and ALDH(br) CD34(neg) UCB cells were characterized within a variety of in vivo and in vitro assays. Primitive progenitors capable of multilineage development were monitored in long- and short-term repopulation assays performed on nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice, and in primary and secondary long-term culture assays. These progenitors were highly enriched within the ALDH(br) CD34+ fraction. This cell fraction also enriched short-term myeloid progenitors that were detected in vitro. By comparison, ALDH(neg) CD34+ cells contained few primitive progenitors and had diminished short-term myeloid potential but exhibited enhanced short-term natural killer (NK) cell development in vitro. The ALDH(br) CD34(neg) cells were not efficiently supported by any of the assays used. These studies suggested that in particular the expression of ALDH delineated distinct CD34+ stem cell and progenitor compartments. The differential expression of ALDH may provide a means to explore normal and malignant processes associated with myeloid and lymphoid development.

Mobilized peripheral blood SSCloALDHbr cells have the phenotypic and functional properties of primitive haematopoietic cells and their number correlates with engraftment following autologous transplantation.

We have developed an approach for identifying primitive mobilized peripheral blood cells (PBSC) that express high levels of aldehyde dehydrogenase (ALDH). PBSC were stained with a fluorescent ALDH substrate, termed BODIPY trade mark -aminoacetaldehyde (BAAA), and then analysed using flow cytometry. A population of cells with a low side scatter (SSC) and a high level of BAAA staining, termed the SSCloALDHbr population, was readily discriminated and comprised a mean of 3 +/- 5% of leukapheresis samples. A mean of 73 +/- 11% of the SSCloALDHbr population expressed CD34 and 56 +/- 25% of all the mobilized CD34+ cells resided within the SSCloALDHbr population. The SSCloALDHbr population was largely depleted of cells with mature phenotypes and enriched for cells with immature phenotypes. Sorted SSCloALDHbr and SSCloALDHbr CD34+ PBSC were enriched for progenitors with the ability to (1) generate colony-forming units (CFU) and long-term culture (LTC)-derived CFU, (2) expand in primary and secondary LTC, and (3) generate multiple cell lineages. In 21 cancer patients who had undergone autologous PBSC transplantation, the number of infused SSCloALDHbr cells/kg highly correlated with the time to neutrophil and platelet engraftment (P < 0.015 and P < 0.003 respectively). In summary, peripheral blood SSCloALDHbr cells have the phenotypic and functional properties of primitive haematopoietic cells and their number correlates with engraftment following autologous transplantation.

Variant forms of alpha-fetoprotein transcripts expressed in human hematopoietic progenitors. Implications for their developmental potential towards endoderm.

Hematopoietic stem cells have been identified as multipotent cells that give rise to all adult hematopoietic lineages. Although the hematopoietic lineage is derived from the mesodermal germ layer in the embryo, recent data suggest that bone marrow cells with an antigenic profile consistent with that of hematopoietic stem cells can also differentiate to cell types of the endodermal lineages, such as hepatocytes. However, the molecular mechanisms associated with these events are entirely unknown. For decades, alpha-fetoprotein (AFP) has been used as a differentiation marker for endodermal cells, because it was thought that the transcription of AFP mRNA is tightly regulated in a developmental and tissue-specific process. In this report we describe two new variant forms of AFP transcripts in human hematopoietic progenitors that are not expressed in mature cells. The variant AFP (vAFP) cDNA sequences isolated from a multipotent hematopoietic cell line, K562, revealed that the vAFP differed from the authentic transcript, consisting of 15 exons, by replacing exon 1 of AFP with one or two exons located in the 5'-untranslated region of the AFP gene. In addition to the K562 cell line, vAFP transcripts were detected in normal bone marrow, thymus, and brain but were not detected in normal spleen, intestine, liver, or the hepatocellular carcinoma cell line, HepG2. This suggests expression in normal hematopoietic progenitors. This hypothesis was confirmed by the finding that CD34(+)Lin(-) hematopoietic progenitor cells purified from cord blood by flow cytometric sorting also expressed the variant transcripts. These results suggest that some hematopoietic progenitors are in a state that permits them to express certain types of transcripts that have been considered unique to endoderm.