Transgenic overexpression of human IL-17E results in eosinophilia, B-lymphocyte hyperplasia, and altered antibody production

We have identified and cloned a novel human cytokine with homology to cytokines of the interleukin-17 (IL-17) family, which we have termed human IL-17E (hIL-17E). With the identification of several IL-17 family members, it is critical to understand the in vivo function of these molecules. We have generated transgenic mice overexpressing hIL-17E using an apolipoprotein E (ApoE) hepatic promoter. These mice displayed changes in the peripheral blood, particularly, a 3-fold increase in total leukocytes consisting of increases in eosinophils, lymphocytes, and neutrophils. Splenomegaly and lymphoadenopathy were predominant and included marked eosinophil infiltrates and lymphoid hyperplasia. CCR3+ eosinophils increased in the blood and lymph nodes of the transgenic mice by 50- and 300-fold, respectively. Eosinophils also increased 8- to 18-fold in the bone marrow and spleen, respectively. In the bone marrow, most of the eosinophils had an immature appearance. CD19+ B cells increased 2- to 5-fold in the peripheral blood, 2-fold in the spleen, and 10-fold in the lymph nodes of transgenic mice, whereas CD4+ T lymphocytes increased 2-fold in both blood and spleen. High serum levels of the cytokines IL-2, IL-4, IL-5, granulocyte colony-stimulating factor, eotaxin, and interferon γ were observed. Consistent with B-lymphocyte increases, serum immunoglobulin (Ig) M, IgG, and IgE were significantly elevated. Antigenic challenge of the transgenic mice with keyhole limpet hemocyanin (KLH) resulted in a decrease in anti-KLH IgG accompanied by increases of anti-KLH IgA and IgE. In situ hybridization of transgenic tissues revealed that IL-17Rh1 (IL-17BR/Evi27), a receptor that binds IL-17E, is up-regulated. Taken together, these data indicate that IL-17E regulates hematopoietic and immune functions, stimulating the development of eosinophils and B lymphocytes. The fact that hIL-17E overexpression results in high levels of circulating eosinophils, IL-4, IL-5, eotaxin, and IgE suggests that IL-17E may be a proinflammatory cytokine favoring Th2-type immune responses.

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Eosinophilia in transgenic mice expressing interleukin 5.

Journal of Experimental Medicine ◽

10.1084/jem.172.5.1425 ◽

1990 ◽

Vol 172 (5) ◽

pp. 1425-1431 ◽

Cited By ~ 392

Author(s):

L A Dent ◽

M Strath ◽

A L Mellor ◽

C J Sanderson

Keyword(s):

Bone Marrow ◽

Transgenic Mice ◽

Lymph Nodes ◽

Peyer's Patches ◽

Peyer’S Patches ◽

Mesenteric Lymph Nodes ◽

Gene Encoding ◽

Interleukin 5

Experiments in vitro suggest that although interleukin 5 (IL-5) stimulates the late stages of eosinophil differentiation, other cytokines are required for the generation of eosinophil progenitor cells. In this study transgenic mice constitutively expressing the IL-5 gene were established using a genomic fragment of the IL-5 gene coupled to the dominant control region from the gene encoding human CD2. Four independent eosinophilic transgenic lines have thus far been established, two of which with 8 and 49 transgene copies, are described in detail. These mice appeared macroscopically normal apart from splenomegaly. Eosinophils were at least 65- and 265-fold higher in blood from transgenics, relative to normal littermates, and approximately two- or sevenfold more numerous relative to blood from mice infected with the helminth Mesocestoides corti. Much more modest increases in blood neutrophil, lymphocyte, and monocyte numbers were noted in transgenics, relative to normal littermates (less than threefold). Thus IL-5 in vivo is relatively specific for the eosinophil lineage. Large numbers of eosinophils were present in spleen, bone marrow, and peritoneal exudate, and were highest in the line with the greatest transgene copy number. Eosinophilia was also noted in histological sections of transgenic lungs, Peyer's patches, mesenteric lymph nodes, and gut lamina propria but not in other tissues examined. IL-5 was detected in the sera of transgenics at levels comparable to those seen in sera from parasite-infected animals. IL-3 and granulocyte/macrophage colony-stimulating factor (GM-CSF) were not found. IL-5 mRNA was detected in transgenic thymus, Peyer's patches, and superficial lymph nodes, but not in heart, liver, brain, or skeletal muscle or in any tissues from nontransgenics. Bone marrow from transgenic mice was rich in IL-5-dependent eosinophil precursors. These data indicate that induction of the IL-5 gene is sufficient for production of eosinophilia, and that IL-5 can induce the full pathway of eosinophil differentiation. IL-5 may therefore not be restricted in action to the later stages of eosinophil differentiation, as suggested by earlier in vitro studies.

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Direct in vivo evidence for increased proliferation of CLL cells in lymph nodes compared to bone marrow and peripheral blood

Leukemia ◽

10.1038/leu.2017.11 ◽

2017 ◽

Vol 31 (6) ◽

pp. 1340-1347 ◽

Cited By ~ 37

Author(s):

T M Herndon ◽

S-S Chen ◽

N S Saba ◽

J Valdez ◽

C Emson ◽

...

Keyword(s):

Bone Marrow ◽

Lymph Nodes ◽

Peripheral Blood

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Impaired Lentiviral Transgene Expression In Vivo Caused by Massive Methylation of SFFV Promoter Sequences.

Blood ◽

10.1182/blood.v116.21.3760.3760 ◽

2010 ◽

Vol 116 (21) ◽

pp. 3760-3760 ◽

Cited By ~ 1

Author(s):

Friederike Herbst ◽

Claudia R Ball ◽

Francesca Tuorto ◽

Wei Wang ◽

Ulrich Kloz ◽

...

Keyword(s):

Bone Marrow ◽

Transgenic Mice ◽

Peripheral Blood ◽

Transgene Expression ◽

Es Cells ◽

Methylation Status ◽

Lentiviral Vectors ◽

Control Vector ◽

Lentiviral Integration

Abstract Abstract 3760 Lentiviral vectors (LV) assure stable transgene expression in vivo, allowing to investigate genes and their functions. In recent years, lentiviral gene transfer was considered to facilitate the generation of transgenic mice with a higher yield of transgenic offspring as compared to commonly used DNA microinjection. We applied LV to generate a mouse model transgenic for SETBP1 and eGFP. Murine zygotes were infected at dE0.5 with lentiviral particles directly injected into the perivitelline space. Specific PCRs for either the SETBP1 transgene or for the WPRE element of the lentiviral construct verified complete lentiviral integration in newborn pups (F0). Lentiviral integration sites were detected using highly sensitive LAM-PCR in 65% of 31 analyzed F0 mice. Germline transmission was shown in a total of 33% vector positive offspring from 5 out of 9 F0 mice. However, no ectopic transcription and overexpression of neither SETBP1 nor eGFP could be detected in transgenic mice. We therefore analyzed the methylation status of the internal SFFV promoter (SFFVp) by bisulfite sequencing. Extensive methylation (around 90%) could be assessed in 18 of 18 analyzed CpGs within the promoter region in F0 animals and in all progeny determined (n=12). We transduced mES cells with LV.SFFV.Setbp1.IRES.eGFP or the corresponding eGFP-expressing control vector to exclude transgene effects on epigenetic silencing of SFFVp sequences in self-inactivating LVs. Differentiation of ES cells infected with the transgene vector and SFFV driven control vector led to a 1.8 – 3.5 fold decrease of eGFP expression. To analyze whether methylation of SFFVp sequences is a common event even in adult tissues, we analyzed the methylation status of peripheral blood in mice transplanted with bone marrow cells transduced with either gammaretroviral vectors (RV) or LV 3 months after transplantation (n=7). Interestingly, SFFVp sequences in peripheral blood of mice transplanted with LV transduced bone marrow were stronger methylated than CpGs of SFFVp in RV transplants. Our data demonstrate that the commonly used SFFV promotor is highly methylated with remarkable strength and frequency during development in vivo and differentiation in vitro. We conclude that lentiviral vectors using an internal SFFV promoter are not suitable for the generation of transgenic mice or constitutive expression studies in hematopoietic cells. Disclosures: No relevant conflicts of interest to declare.

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Mutant U2AF1 Expression Alters Hematopoiesis and Pre-mRNA Splicing in Transgenic Mice

Blood ◽

10.1182/blood.v124.21.827.827 ◽

2014 ◽

Vol 124 (21) ◽

pp. 827-827

Author(s):

Cara Lunn Shirai ◽

James N Ley ◽

Brian S. White ◽

Justin Tibbitts ◽

Jin Shao ◽

...

Keyword(s):

Bone Marrow ◽

Flow Cytometry ◽

Stem Cell ◽

Transgenic Mice ◽

Rna Sequencing ◽

Peripheral Blood ◽

Mrna Splicing ◽

Splice Acceptor Site ◽

Isoform Expression

Abstract Our group and others discovered recurrent heterozygous missense mutations in U2AF1 in 11% of patients with myelodysplastic syndromes (MDS). The U2AF1 gene encodes a splicing factor involved in intronic 3’-splice site recognition, which suggests that perturbations in pre-mRNA splicing play a role in MDS pathogenesis. To study the effects of the most common U2AF1 mutation, U2AF1(S34F), on hematopoiesis and pre-mRNA splicing in vivo, we created site-specific, single-copy, doxycycline-inducible U2AF1(WT) and U2AF1(S34F) transgenic mice. To examine the cell-autonomous effects of mutant U2AF1(S34F), we transplanted transgenic donor bone marrow into wild type recipient mice prior to induction of transgene expression. Following 4 weeks of transgene induction, U2AF1(S34F)-recipient mice have reduced total WBCs in the peripheral blood compared to U2AF1(WT)- and rtTA only-recipient controls (4.3 vs 7.11 and 7.13 K/µl, respectively, p≤0.01), but no significant changes in bone marrow cellularity or spleen size (n=9-11). U2AF1(S34F)-recipient mice have a perturbed mature cell lineage distribution, including reduced monocytes and B cells in both peripheral blood (p≤0.05) and bone marrow (p≤0.01) when compared to control mice (n=9-11). Reduction of bone marrow monocytes occurs as early as 5 days and is associated with increased Annexin V+ (p≤0.05) and phospho-H2AX (p≤0.05) compared to controls, suggesting loss of these cells may be due to apoptosis. In addition, U2AF1(S34F)-recipient mice have increased numbers of progenitors in both bone marrow and spleen by CFU-C methylcellulose assay and flow cytometry for c-Kit+/Lineage- cells, as well as common myeloid progenitors (CMPs), when compared to U2AF1(WT) and rtTA only controls (p≤0.05, n=5-10). U2AF1(S34F)-recipient mice also have an increase in the frequency of bone marrow hematopoietic stem cells (HSCs) measured by flow cytometry for bone marrow KLS (c-Kit+/Lineage-/Sca-1+) cells (p≤0.05). The increase in bone marrow KLS cells in U2AF1(S34F)-recipient mice is seen as early as 5 days and is associated with higher levels of intracellular Ki67 (a marker of cell proliferation) in KLS cells compared to U2AF1(WT) controls (p<0.05, n=8-13). Competitive repopulation studies show a disadvantage for bone marrow cells expressing mutant U2AF1(S34F) compared to U2AF1(WT) at ≥4 months post-transplant in both primary and secondary transplant recipient mice (p≤0.05, n=3-12), suggesting that the increase in KLS cell cycling following U2AF1(S34F) expression may lead to stem cell exhaustion. Collectively, these data indicate U2AF1(S34F) expression alters hematopoiesis in vivo. Next, we performed unbiased RNA sequencing on sorted bone marrow CMPs following 5 days of transgene induction in U2AF1(S34F)- and U2AF1(WT)-transplanted mice (n=3 each). We identified 460 splicing junctions that were differentially expressed in U2AF1(S34F) samples compared to U2AF1(WT) controls (FDR <5%). We observed a preference of the mutant U2AF1(S34F) to skip exons (p=1.3e-05, n=72) and alternative splice sites (p=0.014, n=45) with a T in the -3 position relative to the AG splice acceptor site of differentially-spliced genes; this effect has been previously reported in AML patient samples with U2AF1 mutations. To prioritize altered junctions for further analysis, we intersected mouse CMP junction results with RNA sequencing data from AML patient samples with and without U2AF1 mutations and primary human CD34+ cells over-expressing U2AF1(S34F) or U2AF1(WT). Across species and present in all 3 datasets, we identified homologous dysregulated junctions in 2 genes known to be involved in cancer and stem cell biology: H2AFY and MED24. We validated concordant changes in both H2AFY and MED24 isoform expression by RT-PCR using MDS patient bone marrow samples that have mutant U2AF1(S34F) versus U2AF1(WT) (p<0.001, n=5-6). We are currently testing these isoform changes for their functional contribution to mutant U2AF1-associated phenotypes. Together, these results suggest that mutant U2AF1 expression contributes to the altered hematopoiesis and pre-mRNA splicing observed in patients with U2AF1 mutations. This study also identifies changes in gene isoform expression unique to U2AF1 mutations that may have functional significance for MDS pathogenesis. Disclosures No relevant conflicts of interest to declare.

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Administration of recombinant human interleukin-7 to mice induces the exportation of myeloid progenitor cells from the bone marrow to peripheral sites

Blood ◽

10.1182/blood.v83.2.377.377 ◽

1994 ◽

Vol 83 (2) ◽

pp. 377-385 ◽

Cited By ~ 38

Author(s):

K Grzegorzewski ◽

KL Komschlies ◽

M Mori ◽

K Kaneda ◽

N Usui ◽

...

Keyword(s):

Bone Marrow ◽

Peripheral Blood ◽

White Blood Cells ◽

Intraperitoneal Administration ◽

Fold Increase ◽

Interleukin 7 ◽

Myeloid Progenitor ◽

Fivefold Increase ◽

Myeloid Progenitors

Abstract The administration of recombinant human interleukin-7 (rhIL-7) to mice twice a day for 7 days does not appreciably change bone marrow (BM) cellularity, but does result in a threefold to fivefold increase in the total number of leukocytes in the spleen, an eightfold to 10-fold increase in the total number of nonparenchymal cells (NPC) obtained from the liver, and up to a 20-fold increase in the total number of peripheral white blood cells (WBC). This regimen of rhIL-7 administration also causes a profound reduction in the total number of progenitors in the BM for both single-lineage colony-forming units- culture (CFU-c) (> 90%) and multilineage CFU-granulocyte, erythroid, monocyte, megakaryocyte (CFU-GEMM) (> 99%) colonies. In contrast, mice treated with rhIL-7 exhibited increases in both CFU-c (20- to 40-fold, 20-fold, and 15- to 40-fold) and CFU-GEMM (8- to 10-fold, 30-fold, and 6- to 10-fold) cultured from the peripheral blood, spleen, and NPC, respectively. The increase in CFU in the NPC was accompanied by a fivefold increase in the number of MAC-1+ cells and a ninefold increase in the number of 8C5bright+ cells. Splenectomy of mice before the administration of rhIL-7 further increased the total number of WBC, NPC, and myeloid progenitors as compared with the rhIL-7-treated nonsplenectomized mice. Finally, selective depletion of the BM by intraperitoneal administration of 89Sr (98% reduction in BM cellularity and > 99% reduction in BM myeloid progenitors) abrogated the rhIL-7- induced increases in cellularity and myeloid progenitor number in the peripheral blood, spleen, and NPC. These results show that the changes in myelopoiesis observed after in vivo administration of rhIL-7 to mice result largely from the emigration of myeloid progenitors from the BM through the blood to the spleen, liver, and, possibly, other peripheral organs.

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Faculty Opinions recommendation of Direct in vivo evidence for increased proliferation of CLL cells in lymph nodes compared to bone marrow and peripheral blood.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.727194641.793538177 ◽

2017 ◽

Author(s):

Varsha Gandhi ◽

Prithviraj Bose

Keyword(s):

Bone Marrow ◽

Lymph Nodes ◽

Peripheral Blood

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Age-Associated Changes in Bone Marrow-Derived Extracellular Vesicles May Alter Their Effects on Murine Hematopoietic Stem Cell Function

Blood ◽

10.1182/blood-2020-142444 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 37-37

Author(s):

Sicheng Wen ◽

Jill Kreiling ◽

Mark S Dooner ◽

Elaine Papa ◽

Mandy Pereira ◽

...

Keyword(s):

Bone Marrow ◽

Stem Cell ◽

Population Size ◽

Extracellular Vesicles ◽

Peripheral Blood ◽

Natural Aging ◽

Fold Increase ◽

Vehicle Control ◽

Hematopoietic Stem

Extracellular vesicles (EVs) are critical mediators of intercellular communication within the bone marrow niche and have been implicated in numerous features of aging. However, their role in natural hematopoietic stem cell (HSC) aging has not been fully elucidated. The goal of this work was to test the hypothesis that EVs from whole bone marrow (BM-EVs) can modulate the HSC aging phenotype in vivo. With respect to HSC aging, our prior work showed that, in contrast to the well-known reduced functional capacity and prominent myeloid skewing displayed by old immunophenotypically-defined HSCs, old unseparated whole bone marrow (WBM) (24-26-mo-old) had a 4-fold increase in functional HSCs compared to young (6-8-wk-old) WBM in limit dilution competitive bone marrow transplantation, and showed minimal to no myeloid skewing. In order to test the ability of BM-EVs derived from this total WBM population to alter HSC aging phenotype, we first isolated EVs from WBM flushed from old (24-26-month old) and young (6-8-week old) C57/BL6 (CD45.2) mice by differential centrifugation (2000 × g for 30 min, supernatant centrifuged 100,000 × g for 1 hour, BM-EV pellet collected). Utilizing nanoparticle tracking analysis, we found no difference in mean particle size between old and young BM-EVs, but there was an approximately 2-fold increase in the number of EVs from old WBM compared to young WBM. To test the ability of these BM-EVs to alter HSC function in vivo, we injected old CD45.2 mice with 2 x 109 young BM-EVs and young CD45.2 mice with 2 x 109 old BM-EVs mice via tail vein, daily x 3 days. Control mice were injected with age-matched BM-EVs or vehicle alone. At one-month post injection, we isolated total WBM and Lineage negative/c-Kit+/Sca-1+/CD150+ cells (LSK-SLAM) from the EV-exposed or vehicle control mice. We injected either 3 x 105 WBM cells or 400 LSK-SLAM mixed with 3 x 105 healthy WBM competitor cells from young B6.SJL (CD45.1) mice into lethally irradiated young CD45.1 hosts and measured peripheral blood chimerism and lineage contribution by flow cytometry up to 6 months post-transplant. For the young marrow, exposure to old BM-EVs had no appreciable effects on engraftment capacity or lineage distribution. However, old WBM exposed to young BM-EVs exhibited a significant decrease in engraftment (15% ± 5%) when compared to old WBM exposed to age-matched old BM-EVs (61% ± 14%) or vehicle control (47% ± 7%) (% average donor chimerism ± SEM, n=4-5 mice/group, p<0.04). Similarly, there was a trend toward decreased engraftment capacity by old LSK-SLAM after young BM-EV exposure (7% ± 4%) and increased engraftment capacity by the old LSK-SLAM after old BM-EV exposure (27% ± 10%) compared to vehicle control (15% ± 2%) (% average donor chimerism ± SEM, n=4-5 mice/group, p=not significant (ns)). These results are consistent with our prior data discussed above in which old un-separated WBM, the source of the old BM-EVs, had increased engraftment capacity compared to young WBM. Interestingly, there was also a trend toward reversal of classic myeloid skewing when old LSK-SLAM were exposed to young BM-EVs (36% ± 11%) compared to vehicle control (64% ± 3%) (average myeloid % of donor-derived peripheral blood ± SEM, n=4-5mice/group, p=ns). In addition, consistent with the known increase in LSK-SLAM with age, our preliminary data showed that old mice exposed to young BM-EVs had an approximately 7-fold decrease in the number of LSK-SLAM in marrow, indicating that BM-EVs within the young bone marrow microenvironment may modulate HSC population size. Finally, microRNA (miR) expression profiling (NanoString Technologies) indicated that a number of miRs known to be involved in hematopoiesis, proliferation, self-renewal, differentiation, senescence and inflammation were differentially represented in old and young BM-EVs, with miR-29a, miR-24, and miR-21 significantly increased and miR-105 significantly decreased in old BM-EVs compared to young BM-EVs. Together, these data indicate that young BM-EVs, via transfer of differentially age-related cargo, may alter engraftment capacity, modify the lineage commitment and may regulate LSK-SLAM population size during natural aging. Future studies more clearly delineating age-associated BM-EV effects on HSCs and defining the molecular mechanisms underlying these effects could yield key insights into the natural aging of HSCs and facilitate restoration of healthy hematopoiesis in the elderly. Disclosures No relevant conflicts of interest to declare.

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Administration of recombinant human interleukin-7 to mice induces the exportation of myeloid progenitor cells from the bone marrow to peripheral sites

Blood ◽

10.1182/blood.v83.2.377.bloodjournal832377 ◽

1994 ◽

Vol 83 (2) ◽

pp. 377-385 ◽

Cited By ~ 1

Author(s):

K Grzegorzewski ◽

KL Komschlies ◽

M Mori ◽

K Kaneda ◽

N Usui ◽

...

Keyword(s):

Bone Marrow ◽

Peripheral Blood ◽

White Blood Cells ◽

Intraperitoneal Administration ◽

Fold Increase ◽

Interleukin 7 ◽

Myeloid Progenitor ◽

Fivefold Increase ◽

Myeloid Progenitors

The administration of recombinant human interleukin-7 (rhIL-7) to mice twice a day for 7 days does not appreciably change bone marrow (BM) cellularity, but does result in a threefold to fivefold increase in the total number of leukocytes in the spleen, an eightfold to 10-fold increase in the total number of nonparenchymal cells (NPC) obtained from the liver, and up to a 20-fold increase in the total number of peripheral white blood cells (WBC). This regimen of rhIL-7 administration also causes a profound reduction in the total number of progenitors in the BM for both single-lineage colony-forming units- culture (CFU-c) (> 90%) and multilineage CFU-granulocyte, erythroid, monocyte, megakaryocyte (CFU-GEMM) (> 99%) colonies. In contrast, mice treated with rhIL-7 exhibited increases in both CFU-c (20- to 40-fold, 20-fold, and 15- to 40-fold) and CFU-GEMM (8- to 10-fold, 30-fold, and 6- to 10-fold) cultured from the peripheral blood, spleen, and NPC, respectively. The increase in CFU in the NPC was accompanied by a fivefold increase in the number of MAC-1+ cells and a ninefold increase in the number of 8C5bright+ cells. Splenectomy of mice before the administration of rhIL-7 further increased the total number of WBC, NPC, and myeloid progenitors as compared with the rhIL-7-treated nonsplenectomized mice. Finally, selective depletion of the BM by intraperitoneal administration of 89Sr (98% reduction in BM cellularity and > 99% reduction in BM myeloid progenitors) abrogated the rhIL-7- induced increases in cellularity and myeloid progenitor number in the peripheral blood, spleen, and NPC. These results show that the changes in myelopoiesis observed after in vivo administration of rhIL-7 to mice result largely from the emigration of myeloid progenitors from the BM through the blood to the spleen, liver, and, possibly, other peripheral organs.

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Primitive Human Hematopoietic Cells Are Enriched in Cord Blood Compared With Adult Bone Marrow or Mobilized Peripheral Blood as Measured by the Quantitative In Vivo SCID-Repopulating Cell Assay

Blood ◽

10.1182/blood.v89.11.3919 ◽

1997 ◽

Vol 89 (11) ◽

pp. 3919-3924 ◽

Cited By ~ 328

Author(s):

Jean C.Y. Wang ◽

Monica Doedens ◽

John E. Dick

Keyword(s):

Bone Marrow ◽

Cord Blood ◽

Peripheral Blood ◽

Hematopoietic Cells ◽

Allogeneic Transplantation ◽

Functional Assay ◽

Hematopoietic Stem ◽

Mobilized Peripheral Blood

Abstract We have previously reported the development of in vivo functional assays for primitive human hematopoietic cells based on their ability to repopulate the bone marrow (BM) of severe combined immunodeficient (SCID) and nonobese diabetic/SCID (NOD/SCID) mice following intravenous transplantation. Accumulated data from gene marking and cell purification experiments indicate that the engrafting cells (defined as SCID-repopulating cells or SRC) are biologically distinct from and more primitive than most cells that can be assayed in vitro. Here we demonstrate through limiting dilution analysis that the NOD/SCID xenotransplant model provides a quantitative assay for SRC. Using this assay, the frequency of SRC in cord blood (CB) was found to be 1 in 9.3 × 105 cells. This was significantly higher than the frequency of 1 SRC in 3.0 × 106 adult BM cells or 1 in 6.0 × 106 mobilized peripheral blood (PB) cells from normal donors. Mice transplanted with limiting numbers of SRC were engrafted with both lymphoid and multilineage myeloid human cells. This functional assay is currently the only available method for quantitative analysis of human hematopoietic cells with repopulating capacity. Both CB and mobilized PB are increasingly being used as alternative sources of hematopoietic stem cells in allogeneic transplantation. Thus, the findings reported here will have important clinical as well as biologic implications.

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