Mitochondrial DNA (mtDNA) Sequence Heterogeneity among and within Single Human CD34 Cells, T Cells, B Cells and Granulocytes.

Abstract Abnormalities of mitochondrial DNA (mtDNA) are responsible for a variety of inherited syndromes and have been broadly implicated in aging, cancer, and autoimmunity diseases. Mutations in mtDNA have been reported in myelodysplasia and leukemia, although their pathogenic mechanism remains uncertain. We have described age-dependent accumulation of mtDNA mutations, leading to a high degree of mtDNA sequence heterogeneity among normal marrow and blood CD34 cells as well as in granulocytes (Shin M et al, Blood101:3118 [2003], 103:553 [2004], 103:4466 [2004]). In order to examine mtDNA heterogeneity in detail, we developed a method for analysis of the mtDNA control region from single cells that were sorted by flow cytometry. Highly purified populations of CD34 cells, T cells, B cells, and granulocytes were obtained from five healthy adult donors. The sequence of the individual cells’ mtDNA was compared to the aggregate mtDNA for the respective cell type and differences were expressed as a measure of mtDNA heterogeneity among cells. Overall, heterogeneity was high: for circulating CD34 cells, 38±3.4%; for T cells, 37±14%; B cells, 36±10.8%; and for granulocytes, 48±7.2% (the value for granulocytes statistically differed from CD34 cells; p = 0.03). Most intercellular heterogeneity was due to polyC tract length variability; however, mtDNA base substitution mutations were also prevalent: 15±5.5% in CD34 cells; 15±9.0% in T cells, 15±6.7% in B cells; and 33±2.4% for granulocytes (granulocytes were significantly higher than other cells; p < 0.01). The higher rate of base substitution in granulocytes may reflect their greater exposure to reactive oxygen species. Surprisingly, for both polyC tract length differences and point mutations, the specific mtDNA abnormalities and the proportion of circulating cells characterized by these changes were similar among different cell lineages and relatively constant over time (~2 years) in the same donors. One inference from these results is that mtDNA heterogeneity during development is fixed in the primitive lymphohematopoietic stem cell compartment. In contrast to normal adults, circulating CD34 cells from patients obtained even years after successful allogeneic stem cell transplantation showed a remarkable level of mtDNA homogeneity, similar to the uniformity we have previously observed in cord blood CD34 cells and consistent with limited numbers of stem cells active in these individuals. Leukemic blast cells (from patients with AML-M2, AML evolving from CMML, and T-PLL) also showed a high degree of homogeneity. We propose that mtDNA sequence of single cells may be utilized as a natural genetic marker of hematopoietic progenitors and stem cells; to detect minimal residual disease in leukemia; and as a measure of the accumulation of mutagenic events in mammalian cells in vivo and in vitro.

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Mitochondrial DNA spectra of single human CD34+ cells, T cells, B cells, and granulocytes

Blood ◽

10.1182/blood-2005-01-0150 ◽

2005 ◽

Vol 106 (9) ◽

pp. 3271-3284 ◽

Cited By ~ 15

Author(s):

Yoji Ogasawara ◽

Kazutaka Nakayama ◽

Magdalena Tarnowka ◽

J. Philip McCoy ◽

Sachiko Kajigaya ◽

...

Keyword(s):

T Cells ◽

Mitochondrial Dna ◽

B Cells ◽

Single Cells ◽

Point Mutations ◽

Isolated Cells ◽

Mtdna Mutations ◽

Human Cd34 ◽

Cd34 Cells ◽

Mtdna Sequence

Abstract Previously, we described the age-dependent accumulation of mitochondrial DNA (mtDNA) mutations, leading to a high degree of mtDNA heterogeneity among normal marrow and blood CD34+ clones and in granulocytes. We established a method for sequence analysis of single cells. We show marked, distinct mtDNA heterogeneity from corresponding aggregate sequences in isolated cells of 5 healthy adult donors—37.9% ± 3.6% heterogeneity in circulating CD34+ cells, 36.4% ± 14.1% in T cells, 36.0% ± 10.7% in B cells, and 47.7% ± 7.4% in granulocytes. Most heterogeneity was caused by poly-C tract variability; however, base substitutions were also prevalent, as follows: 14.7% ± 5.7% in CD34+ cells, 15.2% ± 9.0% in T cells, 15.4% ± 6.7% in B cells, and 32.3% ± 2.4% in granulocytes. Many poly-C tract length differences and specific point mutations seen in these same donors but assayed 2 years earlier were still present in the new CD34+ samples. Additionally, specific poly-C tract differences and point mutations were frequently shared among cells of the lymphoid and myeloid lineages. Secular stability and lineage sharing of mtDNA sequence variability suggest that mutations arise in the lymphohematopoietic stem cell compartment and that these changes may be used as a natural genetic marker to estimate the number of active stem cells.

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Human IPS Cells Generated From Adult Peripheral Blood Cells and Purified CD34+ Cells by a Non-Integrating Plasmid.

Blood ◽

10.1182/blood.v116.21.1589.1589 ◽

2010 ◽

Vol 116 (21) ◽

pp. 1589-1589 ◽

Cited By ~ 1

Author(s):

Xiaosong Huang ◽

Bin-Kuan Chou ◽

Prashant Mali ◽

Zhaohui Ye ◽

Sarah N Dowey ◽

...

Keyword(s):

Stem Cells ◽

T Cells ◽

B Cells ◽

Peripheral Blood ◽

Blood Cells ◽

Disease Modeling ◽

Retroviral Vectors ◽

Cd34 Cells ◽

Episomal Vectors ◽

Adult Peripheral Blood

Abstract Abstract 1589 Human induced pluripotent stem cells (iPSCs) that are functionally similar to embryonic stem cells (ESCs) hold great potential for cell and gene therapies, disease modeling and drug development. The earliest success was achieved by using adherent fibroblastic cells and retroviral vectors that transduce fibroblasts very efficiently. It is also highly desirable to reprogram postnatal blood cells, including those from cord blood (CB) and adult peripheral blood (PB), which are easily accessible and less exposed to environmental mutagens. In 2009, we and others have achieved the reprogramming of human postnatal blood cells using the 4 Yamanaka factors delivered by retroviral vectors. We also found that reprogramming efficiencies of CB and PB CD34+ cells are higher than age-matched fibroblasts or MSCs. This may result from an epigenetic profile of hematopoietic CD34+ cells that appears closer to iPSCs/ESCs than that of fibroblasts/MSCs to iPSCs/ESCs. To generate integration-free iPSCs that produce hematopoietic progeny efficiently, we attempted to reprogram adult PB as well as CB cells by OriP/EBNA1 episomal vectors, which were used previously to reprogram foreskin fibroblasts albeit at a low efficiency (Yu/Thomson, 2009). When one of the best combinations (#6, 3 plasmids) was used, 1–3 candidate iPSC clones per 1 million cells were obtained as reported (Yu/Thomson, 2009). We and others found that the efficiency of generating iPS clones was even lower with human adult somatic cells by the 3 vectors. To improve, we constructed a new episomal reprogramming vector system using 1–2 OriP/EBNA1 plasmids. One (pEB-C5) expresses 5 factors (OCT4/SOX2/KLF4/Myc/LIN28), and the second expresses SV40 T antigen (Tg). CB and adult PB CD34+ cells were first cultured for 4 days and expanded ≥4-folds. The expanded cells (1 million) were then transfected once by the 1–2 new OriP/EBNA1 plasmids we constructed. Fourteen days later, we obtained on average 250 and 9 TRA-1-60+, iPSC-like colonies from CB and adult PB cells, respectively, when both pCB-C5 and pEB-Tg were used. A single plasmid (pEB-C5) can also generate iPSCs although the efficiency is ∼4-folds lower. Five characterized iPSC lines derived from CB and adult PB CD34+ cells (with or without Tg) are karyotypically normal and pluripotent. After successful reprogramming and expansion, episomal DNA is gradually lost in proliferating iPSCs. After serial expansions for 11–12 passages, vector DNA was undetectable either as episomes or in the genome of the 5 iPSC lines. We next extended this approach to reprogram un-fractionated adult PB mononuclear cells (PBMCs) including those from a sickle cell patient (SCDB003). To achieve better cell proliferation that is critical to iPSC production, we used a culture condition that favors the formation and proliferation of erythroblasts from PBMCs. PBMCs purified by standard Ficoll gradient were cultured in a serum-free condition with cytokines SCF, EPO and IL-3. Although cell death was observed and cell number decreased significantly in the first 4 days, equal or more cells than input were obtained by day 8. The expanded cells morphologically resemble pro-erythroblast cells, and express high-level CD71. Less than 1.5% of them express markers of T cells (CD3, CD2, CD4 and CD8) and B cells (CD19 and CD20). When 2×106 expanded SCDB003 cells (achievable from PBMCs in 1 ml or less PB) were transfected by the 2 OriP/EBNA1 plasmids and reprogrammed in the presence of butyrate, we observed 8 colonies at day 14 that are TRA-1-60+ and iPSC-like. The second plasmid (pEB-Tg) was not essential although it enhanced the efficiency by ∼4 folds. We picked and characterized 3 iPSC-like colonies derived from PBMCs with or without Tg. All of them express pluripotency markers and behave as typical iPSCs. So far we do not have evidence if they are derived from committed T or B cells that somatic mutations altered and rearranged their genomes. We are currently examining karyotypes, in vivo pluripotency, and status of episomal vectors in 3 PBMC-derived iPSCs. As compared to recent studies using viruses that preferentially reprogram human T cells with a rearranged genome, our method of using 1–2 plasmids is virus-free and genomic alteration-free. The ability to obtain integration-free human iPSCs from a few ml PB by 1–2 plasmids will greatly accelerate uses of iPSCs in both research and future clinical applications, epically for blood disease modeling and treatment. Disclosures: No relevant conflicts of interest to declare.

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Mitochondrial DNA sequence heterogeneity in circulating normal human CD34 cells and granulocytes

Blood ◽

10.1182/blood-2003-11-3949 ◽

2004 ◽

Vol 103 (12) ◽

pp. 4466-4477 ◽

Cited By ~ 24

Author(s):

Myung Geun Shin ◽

Sachiko Kajigaya ◽

Magdalena Tarnowka ◽

J. Philip McCoy ◽

Barbara C. Levin ◽

...

Keyword(s):

Mitochondrial Dna ◽

Residual Disease ◽

Hematologic Malignancies ◽

Source Analysis ◽

Sequence Heterogeneity ◽

Mtdna Mutations ◽

Human Cd34 ◽

Cd34 Cells ◽

Mtdna Sequence ◽

Normal Human

Abstract We have reported marked mitochondrial DNA (mtDNA) sequence heterogeneity among individual CD34 clones from adult bone marrow (BM) and the age-dependent accumulation of mtDNA mutations in this mitotically active tissue. Here, we show direct evidence of clonal expansion of cells containing mtDNA mutations and that the mtDNA sequence may be easily determined by using peripheral blood (PB) as a CD34 cell source. Analysis of 594 circulating CD34 clones showed that 150 (25%) had mtDNA sequences different from the same donor's corresponding aggregate sequence. Examination of single granulocytes indicated that 103 (29%) from the same 6 individuals showed mtDNA heterogeneity, with sequences distinct from the corresponding aggregate tissue sequence and from the sequences of other single granulocytes. Circulating and BM CD34 cells showed virtually identical patterns of mtDNA heterogeneity, and the same changes were seen in progeny granulocytes as in their progenitors, indicating that blood sampling could be used in studies to determine whether mtDNA reflects an individual's cumulative or recent exposure to mutagens; as a marker of individual hematopoietic progenitors, stem cells, and their expansion; and for the detection of minimal residual disease in hematologic malignancies of CD34 cell origin. (Blood. 2004;103:4466-4477)

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Absence of Evidence Implicating Hematopoietic Stem Cells As Common Progenitors for DLBCL Mutations

Blood ◽

10.1182/blood.v128.22.4107.4107 ◽

2016 ◽

Vol 128 (22) ◽

pp. 4107-4107

Author(s):

Max Jan ◽

Florian Scherer ◽

David M. Kurtz ◽

Aaron M Newman ◽

Henning Stehr ◽

...

Keyword(s):

Stem Cells ◽

T Cells ◽

B Cells ◽

Hematopoietic Stem Cells ◽

Research Funding ◽

Phylogenetic Trees ◽

Somatic Mutations ◽

Hematopoietic Stem ◽

Tumor Biopsies ◽

Myd88 L265p

Abstract Background: Pre-leukemic hematopoietic stem cells (HSC) have been implicated in AML (Jan et al STM 2012) and also for several lymphoid leukemias including ALL, HCL, and CLL. Separately, relapse of ALL following CD19 CAR-T cell therapy has been associated with lymphomyeloid lineage switch. Finally, healthy persons with clonally expanded HSCs are at increased risk of hematologic malignancies including lymphomas, and in mouse DLBCL models we previously demonstrated the oncogenic sufficiency of BCL6 overexpression in HSC (Green et al 2014 Nat Comm). Nevertheless, the cellular origin of DLBCL in the majority of patients is not definitively known. We sought to investigate the presence of mutations found in DLBCL within matched HSCs. Methods: We deeply genotyped somatic mutations in diagnostic biopsy tissues of 16 patients with DLBCL using CAPP-Seq to a median sequencing depth of 1100x (Newman et al 2014 Nat Med; Scherer et al 2015 ASH). We then profiled each patient for evidence implicating HSCs using somatic mutation lineage tracing, in either direct or indirect fashion. For direct evaluation, we used highly purified, serially FACS-sorted HSCs from grossly uninvolved bone marrow (BM) (n=5; Fig 1a-b). For indirect assessment, we either profiled serial tumor biopsies (n=13), or interrogated sorted cells from terminally differentiated blood lineages (n=7), including peripheral CD3+ T cells, CD14+ Monocytes, and B cells expressing a light-chain discordant to that of tumor isotype. HSCs and differentiated lineages were then interrogated by direct genotyping, using 3 highly sensitive orthogonal quantitative methods, including Myd88 L265P droplet digital PCR (n=6), BCL6 translocation breakpoint qPCR (n=4), and DLBCL CAPP-Seq profiling of 268 genes (n=5). We used the theoretical limit of detection (LOD) genotyping performance for CAPP-Seq (0.001%, Newman et al 2016 Nat Biotech), and established analytical sensitivity of our custom MYD88 ddPCR via limiting dilution (~1%). These LODs met or exceeded the expected limit of sorting impurity by FACS (~1%). For 6 patients experiencing one or more DLBCL relapse, we deeply profiled 13 serial tumor biopsies by CAPP-Seq, and then assessed overlap in somatic mutations and VDJ sequences in biopsy pairs as additional indirect evidence implicating HSCs. Results: We obtained a median of ~2000 sorted HSCs and ~1700 sorted cells from differentiated lineages, and genotyped each population using one or more of the 3 direct genotyping methods described above. Three patients with sufficient cell numbers were profiled both by CAPP-Seq and either ddPCR (n=2) or qPCR (n=1). Surprisingly, we found no evidence implicating HSCs either directly or indirectly in any of the 16 patients, regardless of the assay employed or the cell types/lineages genotyped (e.g., Fig 1b). In 2 patients with MYD88 L265P mutations, we found evidence for MYD88+ B-cells with discordant light chains by ddPCR (~0.1%) potentially implicating common lymphoid precursors (CLPs), but found no evidence for similar involvement of T-cells or monocytes. In 6 DLBCL patients experiencing relapse, tumor pairs profiled by CAPP-Seq (median depth 957) shared 93% of somatic mutations (75-100%, Fig 1c). Such pairs invariably shared clonal IgH VDJ rearrangements (4/4, 100%), thus implicating a common progenitor arising in later stages of B-cell development, not HSCs. Conclusions: We find no evidence to implicate HSCs in the derivation of DLBCL. While formal demonstration of absence of pre-malignant HSCs in DLBCL would require overcoming practical and technical limitations (including number of available HSCs, sorting purity, and genotyping sensitivity), the pattern of shared somatic alterations at relapse makes this highly unlikely. We speculate that unlike lymphoid leukemias, the cell-of-origin for most DLBCLs reside later in B-lymphopoiesis, beyond CLPs. Figure. (a) HSC sorting from BM by FACS (b) Allele frequencies of mutations found by CAPP-Seq in an examplary DLBCL case (x-axis) compared to the same variants in HSCs (y-axis). (c) Phylogenetic trees of DLBCL patients experiencing relapse (n=6) with tumor pairs sequenced by CAPP-Seq. Shown are the evolutionary distances between (i) germline and common inferrable progenitor (CIP) illustrating the fraction of shared mutations between tumor pairs, and (ii) CIP and both diagnostic (tumor 1) and relapse tumors (tumor 2) indicating unique mutations to each tumor. Figure. (a) HSC sorting from BM by FACS (b) Allele frequencies of mutations found by CAPP-Seq in an examplary DLBCL case (x-axis) compared to the same variants in HSCs (y-axis). (c) Phylogenetic trees of DLBCL patients experiencing relapse (n=6) with tumor pairs sequenced by CAPP-Seq. Shown are the evolutionary distances between (i) germline and common inferrable progenitor (CIP) illustrating the fraction of shared mutations between tumor pairs, and (ii) CIP and both diagnostic (tumor 1) and relapse tumors (tumor 2) indicating unique mutations to each tumor. Disclosures Newman: Roche: Consultancy. Levy:Kite Pharma: Consultancy; Five Prime Therapeutics: Consultancy; Innate Pharma: Consultancy; Beigene: Consultancy; Corvus: Consultancy; Dynavax: Research Funding; Pharmacyclics: Research Funding. Diehn:Novartis: Consultancy; Quanticel Pharmaceuticals: Consultancy; Roche: Consultancy; Varian Medical Systems: Research Funding.

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Effect of Human Mesenchymal Stem Cells on Xenogeneic T and B Cells Isolated from Lupus-Prone MRL.Faslpr Mice

Stem Cells International ◽

10.1155/2020/5617192 ◽

2020 ◽

Vol 2020 ◽

pp. 1-10

Author(s):

Hong Kyung Lee ◽

Eun Young Kim ◽

Hyung Sook Kim ◽

Eun Jae Park ◽

Hye Jin Lee ◽

...

Keyword(s):

Stem Cells ◽

T Cells ◽

Mesenchymal Stem Cells ◽

B Cells ◽

Lupus Erythematosus ◽

Human Mesenchymal Stem Cells ◽

Preclinical Studies ◽

Dependent Manner ◽

T And B Cells

Systemic lupus erythematosus (SLE) is an autoimmune disease, which is characterized by hyperactivation of T and B cells. Human mesenchymal stem cells (hMSCs) ameliorate the progression of SLE in preclinical studies using lupus-prone MRL.Faslpr mice. However, whether hMSCs inhibit the functions of xenogeneic mouse T and B cells is not clear. To address this issue, we examined the in vitro effects of hMSCs on T and B cells isolated from MRL.Faslpr mice. Naïve hMSCs inhibited the functions of T cells but not B cells. hMSCs preconditioned with IFN-γ (i) inhibited the proliferation of and IgM production by B cells, (ii) attracted B cells for cell–cell interactions in a CXCL10-dependent manner, and (iii) inhibited B cells by producing indoleamine 2,3-dioxygenase. In summary, our data demonstrate that hMSCs exert therapeutic activity in mice in three steps: first, naïve hMSCs inhibit the functions of T cells, hMSCs are then activated by IFN-γ, and finally, they inhibit B cells.

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A Detailed Phenotypic Analysis Using Six Color Flow Cytometry of Lymphocyte Subsets Mobilized with AMD3100 Compared to G-CSF.

Blood ◽

10.1182/blood.v104.11.408.408 ◽

2004 ◽

Vol 104 (11) ◽

pp. 408-408 ◽

Cited By ~ 1

Author(s):

Yoshiyuki Takahashi ◽

S. Chakrabarti ◽

R. Sriniivasan ◽

A. Lundqvist ◽

E.J. Read ◽

...

Keyword(s):

Flow Cytometry ◽

T Cells ◽

T Cell ◽

B Cells ◽

Cd8 T Cells ◽

Cd4 T Cells ◽

Lymphocyte Subsets ◽

Phenotypic Analysis ◽

Color Flow ◽

Cd34 Cells

Abstract AMD3100 (AMD) is a bicyclam compound that rapidly mobilizes hematopoietic progenitor cells into circulation by inhibiting stromal cell derived factor-1 binding to its cognate receptor CXCR4 present on CD34+ cells. Preliminary data in healthy donors and cancer patients show large numbers of CD34+ cells are mobilized following a single injection of AMD3100. To determine whether AMD3100 mobilized cells would be suitable for allografting, we performed a detailed phenotypic analysis using 6 color flow cytometry (CYAN Cytometer MLE) of lymphocyte subsets mobilized following the administration of AMD3100, given as a single 240mcg/kg injection either alone (n=4) or in combination with G-CSF (n=2: G-CSF 10 mcg/kg/day x 5: AMD3100 given on day 4). Baseline peripheral blood (PB) was obtained immediately prior to mobilization; in recipients who received both agents, blood was analyzed 4 days following G-CSF administration as well as 12 hours following administration of AMD3100 and a 5th dose of G-CSF. AMD3100 alone significantly increased from baseline the PB WBC count (2.8 fold), Absolute lymphocyte count (ALC: 2.5 fold), absolute monocyte count (AMC: 3.4 fold), and absolute neutrophil count (ANC: 2.8 fold). Subset analysis showed AMD3100 preferentially increased from baseline PB CD34+ progenitor counts (5.8 fold), followed by CD19+ B-cells (3.7 fold), CD14+ monocytes (3.4 fold), CD8+ T-cells (2.5 fold), CD4+ T-cells (1.8 fold), with a smaller increase in CD3−/CD16+ or CD56+ NK cell counts (1.6 fold). There was no change from baseline in the % of CD4+ or CD8+ T-cell expressing CD45RA, CD45RO, or CD56, CD57, CD27, CD71 or HLA-DR. In contrast, there was a decline compared to baseline in the mean percentage of CD3+/CD4+ T-cells expressing CD25 (5.5% vs 14.8%), CD62L (12.1% vs 41.1%), CCR7 (2.1% vs 10.5%) and CXCR4 (0.5% vs 40.9%) after AMD3100 administration; similar declines in expression of the same 4 surface markers were also observed in CD3+/CD8+ T-cells. A synergistic effect on the mobilization of CD34+ progenitors, CD19+ B cells, CD3+ T-cells and CD14+ monocytes occurred when AMD3100 was combined with G-CSF (Figure). In those receiving both AMD3100 and G-CSF, a fall in the % of T-cells expressing CCR7 and CXCR4 occurred 12 hours after the administration of AMD3100 compared to PB collected after 4 days of G-CSF; no other differences in the expression of a variety activation and/or adhesion molecules on T-cell subsets were observed. Whether differences in lymphocyte subsets mobilized with AMD3100 alone or in combination with G-CSF will impact immune reconstitution or other either immune sequela (i.e. GVHD, graft-vs-tumor) associated with allogeneic HCT is currently being assessed in an animal model of allogeneic transplantation.

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Evidence for Efficacy and Safety of Lentiviral Mediated Gene Transfer in T Cells and CD34+ Cells from Wiskott-Aldrich Syndrome Patients.

Blood ◽

10.1182/blood.v108.11.3279.3279 ◽

2006 ◽

Vol 108 (11) ◽

pp. 3279-3279

Author(s):

Samantha Scaramuzza ◽

Sara Trifari ◽

Francesco Marangoni ◽

Silvana Martino ◽

Ayse Metin ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

T Cells ◽

Gene Transfer ◽

Lentiviral Vector ◽

Efficacy And Safety ◽

Wiskott Aldrich Syndrome ◽

Cd34 Cells ◽

Was Gene

Abstract Wiskott-Aldrich Syndrome (WAS) is an X-linked primary immunodeficiency characterized by eczema, recurrent infections, severe hemorrhages and lymphomas. Transplantation of hematopoietic stem cells from HLA-identical sibling donors is a resolutive treatment, but it is available only for a minority of patients. Therapy based on the transplant of genetically correct autologous stem cells could represent a valid alternative approach. We investigated the efficacy and the safety of WAS gene transfer using HIV-based lentiviral vector encoding for WAS cDNA under the control of an autologous promoter (1.6 kb). T cells obtained from WAS patients showed normal level of WAS expression after lentiviral transduction. Transduced T cells showed a correction in TCR-driven proliferation and IL-2 production. Furthermore, a selective growth advantage of transduced T cells was observed in long-term in vitro cultures. Studies in T cell clones generated from transduced WAS CD4+ T cells revealed that 1–2 vector copies were necessary and sufficient to correct T cells function. CD34+ cells, isolated from mobilized peripheral blood and bone marrow of healthy donors, were transduced using WASP or GFP-encoding lentiviral vectors. Cells were cultured in the presence of different cytokines to investigate if WAS gene transfer could have any effect on short and long-term differentiation (CFU-C, LTC-IC and B/NK assays). Transduction resulted in a comparable number of CFU-C and LTC-IC colonies and normal B and NK cells differentiation with respect to untransduced cells. Furthermore, transduction of CD34+ cells isolated from the bone marrow of a WAS patient was performed under optimized culture conditions. Lentiviral gene transfer led to restoration of WASP expression in differentiated cells with copy number ranging from 1 to 5 copies per cell. In conclusion, our data demonstrate that the WAS promoter/cDNA-containing lentiviral vector can efficiently transduce and restore WASP expression in CD34+ cells and T cells from WAS patients. Experiments in the Rag2−/−/γchain- murine model are ongoing to test the efficacy and safety of the WASP transduced CD34+ cells. Together, our studies provide a preclinical basis for the implementation of a gene therapy trial for WAS patients.

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Inability to Express HOXA Cluster and BCL11A Genes Compromises Self-Renewal and Multipotency of hESC-Derived Hematopoietic Cells

Blood ◽

10.1182/blood.v120.21.1190.1190 ◽

2012 ◽

Vol 120 (21) ◽

pp. 1190-1190 ◽

Cited By ~ 1

Author(s):

Diana R Dou ◽

Arazin Minasian ◽

Maria I Sierra ◽

Pamela Saarikoski ◽

Jian Xu ◽

...

Keyword(s):

Stem Cells ◽

B Cells ◽

Proliferative Potential ◽

Pluripotent Cells ◽

Hoxa Cluster ◽

Self Renewal ◽

Hematopoietic Stem ◽

Cd34 Cells ◽

Hoxa Genes

Abstract Abstract 1190 The inability to derive functional hematopoietic stem cells (HSCs) in vitro from pluripotent cells prevents widespread utilization of HSCs in the clinic; however, the molecular defects compromising the in vitro generated hematopoietic stem/progenitor cells (HSPCs) are unknown. Using a two-step differentiation method in which human embryonic stem cells (hESCs) were first differentiated into embryo bodies (EBs) and then CD34+ cells from hEBs were co-cultured on OP9M2 bone marrow mesenchymal stem cell (MSC) stroma (hEB-OP9), we were able to derive HSPCs expressing the HSC immunophenotype (CD34+CD38−CD90+CD45+) (hereafter termed CD90+HSPCs). Colony forming and stroma co-culture assays demonstrated that the hEB-OP9 CD90+HSPCs were able to differentiate into myelo-erythroid lineages and T-cells. However, when comparing CD90+HSPCs from hEB-OP9 to those from fetal liver (FL)—an in vivo source of HSCs—the former remained severely functionally limited in their proliferative potential and ability to differentiate into B-cells. To identify the basis of the proliferative and differentiation defects, we performed microarray analysis to define gene expression differences between CD90+HSPCs derived from hEB-OP9, FL, early 3–5 week placenta (PL) and an earlier stage of hESC differentiation (hEB). This analysis revealed establishment of the general hematopoietic transcription factor network (e.g. SCL, RUNX1, CMYB, ETV6, HOXB4, MYB), demonstrating the successful differentiation and identification of hematopoietic cells using our two-step culturing techniques and immunophenotype criteria. Moreover, evaluation of Spearman coefficients confirmed CD90+HSPCs isolated from hEB-OP9 culture were brought into closer resemblance of the hFL CD90+HSPCs as compared to to the developmentally immature hEB and hPL CD90+HSPCs. Encouragingly, hEB-OP9 CD90+HSPCs displayed downregulation of expression of genes related to hemogenic endothelium development associated with hEB and hPL while genes critical in HSPC function, including DNA repair and chromatin modification, were upregulated to levels comparable to hFL-HSPCs. However, a subgroup of FL HSPC genes could not be induced in hEB-OP9 HSPCs, including the HOXA cluster genes and BCL11A—implicated in HSC self-renewal and B-cell formation, respectively. Interestingly, absence of HOXA genes and BCL11A and poor proliferative potential were also observed in HSPCs from early placenta, suggesting these defects are not in vitro artifacts but instead reflect an inability of hEB-OP9 HSPCs to complete developmental maturation. To validate the necessity of HOXA genes and BCL11A in proliferation potential and multipotency, we next utilized shRNAs to target MLL—the upstream regulator of the HOXA cluster—, individual HOXA genes, or BCL11A in FL-HSPCs to test whether knockdown was sufficient to recapitulate the defects observed in hESC-derived HSPCs. Knockdown of HOXA7 resulted in the loss of CD34+ cells while HOXA9 shRNA-treated cells displayed a loss of more differentiated CD38hi cells. MLL knockdown depleted both CD38+ and CD34+ populations. BCL11A silencing resulted in the loss of B-cells. These studies identify HOXA genes and BCL11A as developmentally regulated genes essential for generating self-renewing, multipotent HSCs from pluripotent cells. Disclosures: No relevant conflicts of interest to declare.

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Co-Stimulatory Blockade With CTLA4-Ig Permits Transplantation Of Human Hematopoietic Stem Cells and HLA Incompatible T Cells In NOD/SCID γ Null (NSG) Mouse Model

Blood ◽

10.1182/blood.v122.21.1999.1999 ◽

2013 ◽

Vol 122 (21) ◽

pp. 1999-1999

Author(s):

Annie L. Oh ◽

Dolores Mahmud ◽

Benedetta Nicolini ◽

Nadim Mahmud ◽

Elisa Bonetti ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

T Cells ◽

Stem Cell ◽

T Cell ◽

Nsg Mice ◽

Human Cd34 ◽

Cd34 Cells

Abstract Our previous studies have shown the ability of human CD34+ cells to stimulate T cell alloproliferative responses in-vitro. Here, we investigated anti-CD34 T cell alloreactivity in-vivo by co-transplanting human CD34+ cells and allogeneic T cells of an incompatible individual into NSG mice. Human CD34+ cells (2x105/animal) were transplanted with allogeneic T cells at different ratios ranging from 1:50 to 1:0.5, or without T cells as a control. No xenogeneic GVHD was detected at 1:1 CD34:T cell ratio. Engraftment of human CD45+ (huCD45+) cells in mice marrow and spleen was analyzed by flow cytometry. Marrow engraftment of huCD45+ cells at 4 or 8 weeks was significantly decreased in mice transplanted with T cells compared to control mice that did not receive T cells. More importantly, transplantation of T cells at CD34:T cell ratios from 1:50 to 1:0.5 resulted in stem cell rejection since >98% huCD45+ cells detected were CD3+. In mice with stem cell rejection, human T cells had a normal CD4:CD8 ratio and CD4+ cells were mostly CD45RA+. The kinetics of human cell engraftment in the bone marrow and spleen was then analyzed in mice transplanted with CD34+ and allogeneic T cells at 1:1 ratio and sacrificed at 1, 2, or 4 weeks. At 2 weeks post transplant, the bone marrow showed CD34-derived myeloid cells, whereas the spleen showed only allo-T cells. At 4 weeks, all myeloid cells had been rejected and only T cells were detected both in the bone marrow and spleen. Based on our previous in-vitro studies showing that T cell alloreactivity against CD34+ cells is mainly due to B7:CD28 costimulatory activation, we injected the mice with CTLA4-Ig (Abatacept, Bristol Myers Squibb, New York, NY) from d-1 to d+28 post transplantation of CD34+ and allogeneic T cells. Treatment of mice with CTLA4-Ig prevented rejection and allowed CD34+ cells to fully engraft the marrow of NSG mice at 4 weeks with an overall 13± 7% engraftment of huCD45+ marrow cells (n=5) which included: 53±9% CD33+ cells, 22±3% CD14+ monocytes, 7±2% CD1c myeloid dendritic cells, and 4±1% CD34+ cells, while CD19+ B cells were only 3±1% and CD3+ T cells were 0.5±1%. We hypothesize that CTLA4-Ig may induce the apoptotic deletion of alloreactive T cells early in the post transplant period although we could not detect T cells in the spleen as early as 7 or 10 days after transplant. Here we demonstrate that costimulatory blockade with CTLA4-Ig at the time of transplant of human CD34+ cells and incompatible allogeneic T cells can prevent T cell mediated rejection. We also show that the NSG model can be utilized to test immunotherapy strategies aimed at engrafting human stem cells across HLA barriers in-vivo. These results will prompt the design of future clinical trials of CD34+ cell transplantation for patients with severe non-malignant disorders, such as sickle cell anemia, thalassemia, immunodeficiencies or aplastic anemia. Disclosures: No relevant conflicts of interest to declare.

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Clonal Tracking of the Geographic Distribution of Hematopoiesis in Nonhuman Primates Provides New Insights into HSPC Migration and Differentiation

Blood ◽

10.1182/blood.v126.23.241.241 ◽

2015 ◽

Vol 126 (23) ◽

pp. 241-241

Author(s):

Chuanfeng Wu ◽

Samson J Koelle ◽

Brian Li ◽

Diego Espinoza ◽

Rong Lu ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

B Cells ◽

Nk Cells ◽

Geographic Distribution ◽

Iliac Crest ◽

T Cell Development ◽

Autologous Transplantation ◽

Post Transplant ◽

Cd34 Cells

Abstract Hematopoietic stem and progenitor cells (HSPC) primarily reside in bone marrow (BM) niches, but are also found in peripheral blood (PB) in small numbers. Large numbers of HSPC can be pharmacologically mobilized from the BM into the PB and home back to BM niches following transplantation. We used the rhesus macaque to study the process of hematopoiesis in both space and time following autologous transplantation, as a model with great relevance to humans. We labeled individual CD34+ HSPC and their progeny with genetic "barcodes" via lentiviral transduction with a very high diversity barcode library, allowing tracking of the output from individual HSPC clones in vivo following autologous transplantation, in a quantitative and sensitive manner (Wu et al., 2014). In this study we investigated the pattern of HSPC clonal output in various anatomic niches, sampling left (L) vs right (R) iliac crest BM, PB and lymph node (LN) over time post ablative autologous transplantation, for all major hematopoietic lineages and CD34+ HSPC. We tracked thousands of HSPC clones in 6 rhesus macaques from 3.5 months (m) to up to 18.5m post transplantation. L and R iliac crest BM and PB were serially collected and CD34+ HSPC along with CD3+CD4+ T, CD3+CD8+ T, CD20+ B, CD14+ monocytes (Mo), CD33+ granulocytes (Gr), and two NK cell subsets (CD3-CD20-CD14-CD16+/ or CD56+) were purified. In all animals, there was marked geographic segregation of CD34+ HSPC for at least 6m post-transplant, with individual clones localized only to the L or R side but not both (Pearson correlation at 3.5-6m: r=0.019±0.08 for CD34+ L vs R, n=6), despite rapid expansion of CD34+ HSPC during early engraftment. This result suggests that during this phase of recovery, HSPC spread contiguously in the BM, and there is little geographic "mixing" via egress into and re-entry from the PB. With time between 7m and 18.5m post-transplant, the distribution of CD34+ HSPC became more homogeneous, with clones detected on both L and R (r=0.52±0.22 at 12-18.5m, n=3). The geographic restriction of clones at earlier time points suggests that release and homing may be dependent on local contiguous niche occupancy status, with migration only occurring following hematopoietic recovery. We next examined the clonal distribution of the more mature lineage-committed cells in the L vs R BM and in PB. Gr, Mo and B cells were produced locally in BM, with the clonal pattern of each lineage matching the CD34+ cells collected from the same side BM time point through 6m (r>0.80 for each lineage vs same side CD34+ cells), and distinct from same lineages (r<0.16 for each lineage in L vs in R BM) and CD34+ cells on the other side (r<-0.01 for each lineage vs other side CD34+ cells). CD16+CD56-/dim NK clones were completely shared between PB and both L and R BM, suggesting they were not produced locally in BM, but instead in other sites with homogenous lodging or homing back to BM. Most surprisingly, we found population of CD3+CD8+ T cells that appeared to be produced locally, with barcodes matching the CD34+ HSPC at the same location, suggesting a novel T cell development pathway within the BM for this subset during early hematopoietic reconstitution. In contrast, CD3+CD4+ BM T cells had similar clonal constitution on the L vs R, and matched the PB, suggesting they had re-circulated back to the BM following maturation elsewhere, such as the thymus. T, B and NK cells from two LNs obtained simultaneously were also analyzed. Clonal contributions to T, B, and NK cells from L vs R LNs were highly correlated (r=0.95, r=0.88, and r=0.89 respectively). The clonal composition of T or B cells in LN were shared with circulating T or B cells (r=0.88, r=0.85 respectively), while LN NK cells (which are primarily CD56+/CD16-) shared barcodes with circulating CD16-CD56+ NK (r=0.81), not with PB CD16+CD56- cells(r=0.26), suggesting a non-precursor/progeny relationship. Our model for the first time documents the dynamics of HSPC geographic distribution and migration in primates following transplantation, findings with direct clinical relevance, and provides new insights into hematopoietic lineage development, including a potential novel T cell development pathway in the BM. Our findings may also help explain the extremely patchy distribution of hematopoiesis in humans following transplantation or in the setting of marrow failure or aging, and suggest that analysis of individual BM samples may not fully reflect ongoing global hematopoiesis. Disclosures No relevant conflicts of interest to declare.

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