scholarly journals Multiple allogeneic progenitors in combination function as a unit to support early transient hematopoiesis in transplantation

2016 ◽  
Vol 213 (9) ◽  
pp. 1865-1880 ◽  
Author(s):  
Takashi Ishida ◽  
Satoshi Takahashi ◽  
Chen-Yi Lai ◽  
Masanori Nojima ◽  
Ryo Yamamoto ◽  
...  
Keyword(s):  
Hematopoietic Cell Transplantation ◽  
Initial Time ◽  
Hematopoietic Stem ◽  
Immunodeficient Mice ◽  
Single Donor ◽  
Hematopoietic Recovery ◽  
Hematopoietic Reconstitution ◽  
Cd34 Cells ◽  
Combination Function ◽  
Donor Source

Cord blood (CB) is a valuable donor source in hematopoietic cell transplantation. However, the initial time to engraftment in CB transplantation (CBT) is often delayed because of low graft cell numbers. This limits the use of CB. To overcome this cell dose barrier, we modeled an insufficient dose CBT setting in lethally irradiated mice and then added hematopoietic stem/progenitor cells (HSCs/HPCs; HSPCs) derived from four mouse allogeneic strains. The mixture of HSPCs rescued recipients and significantly accelerated hematopoietic recovery. Including T cells from one strain favored single-donor chimerism through graft versus graft reactions, with early hematopoietic recovery unaffected. Furthermore, using clinically relevant procedures, we successfully isolated a mixture of CD34+ cells from multiple frozen CB units at one time regardless of HLA-type disparities. These CD34+ cells in combination proved transplantable into immunodeficient mice. This work provides proof of concept that when circumstances require support of hematopoiesis, combined multiple units of allogeneic HSPCs are capable of early hematopoietic reconstitution while allowing single-donor hematopoiesis by a principal graft.

scholarly journals Novel Integrated Autologous Hematopoietic Stem Cell Tracking in Nonhuman Primates Reveals Successive Pattern of Multi-Lineage Reconstitution after Total Body Irradiation

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 2910-2910
Author(s):  
Jennifer E Adair ◽  
Shaina Porter ◽  
Kevin Haworth ◽  
Kenric Tam ◽  
Hans-Peter Kiem ◽  
...  
Keyword(s):  
Stem Cell ◽  
Dna Barcoding ◽  
Total Body Irradiation ◽  
Integration Site ◽  
Hematopoietic Stem ◽  
Hematopoietic Recovery ◽  
Hematopoietic Reconstitution ◽  
Cd34 Cells ◽  
Total Body ◽  
Autologous Hsct

Abstract Hematopoietic stem cell transplantation (HSCT) has been used to cure patients for over four decades, yet the kinetics and clonal dynamics of repopulation by multipotent HSCs remains unresolved. Two sequence-based methods have been used to measure HSC contribution in preclinical models, integration site (IS) analysis and DNA barcoding. Both methods are accomplished by retrovirus tagging of HSCs and each confers different methodological constraints. We hypothesized that IS analysis and DNA barcoding together would provide more robust HSCT reconstitution data if applied to the clinically relevant nonhuman primate autologous HSCT model. We developed a high complexity (~1.2 million), DNA-barcoded LV library encoding the chemotherapy resistance gene MGMT(P140K) and enhanced green fluorescent protein (GFP) to measure hematopoietic reconstitution following total body irradiation (1020 cGy) and autologous HSCT in two pigtailed macaques. In both animals, hematopoietic recovery from TBI was achieved within 1-2 months after HSCT. We observed ~1 and 12% GFP+ peripheral blood white blood cells (PB WBCs) following hematopoietic recovery in these animals, respectively. We performed barcode and IS retrieval from PB WBCs collected early (1 month) after HSCT and observed <0.001% of infused clones contributing to hematopoiesis by DNA barcoding. We identified 42% fewer clones by IS analysis of the same sample, but with similar contributions of the most dominant clones identified by each method. This suggests DNA barcoding provides greater retrieval of low abundance clones. Over 1 year following HSCT, a total of 5,089 (0.4%) and 20,799 (1.7%) infused clones contributed to hematopoiesis in each animal, respectively, with polyclonal contribution patterns. At 1 year post-transplant, we analyzed PB WBC subsets and identified a total of 116 and 2,246 clones respectively contributing to each of three lineages (T, B, and granulocyte), suggesting <0.2% of infused, gene-modified CD34+ cells displayed multi-lineage potential. Some of these clones emerged in the periphery as early as 1 month after HSCT, indicating a successive pattern of multi-lineage clonal behavior. Adjusting for marking levels, we estimate that 1 in 10,000 transplanted CD34+ cells gave rise to trilineage hematopoiesis at 1 year post-HSCT. We then challenged these animals with chemotherapy (O6-benzylguanine and BCNU) to provide gene modified cells with a selective advantage in vivo. We observed moderate myelosuppression following chemotherapy with stable increases in GFP+ PB WBCs to ~2.5 and 22% in each respective animal. Analysis of the clonal repertoire after chemotherapy identified a significant number of new clones not previously identified in the first year after transplant. This suggests that >1 in 10,000 infused CD34+ cells is capable of trilineage hematopoiesis. In sum, the combination of retrovirus integration site analysis with molecular barcode analysis provides synergistic methods in understanding the kinetics and clonal dynamics of hematopoietic reconstitution following autologous HSCT and revealed a successive pattern of multi-lineage clonal behavior. Disclosures No relevant conflicts of interest to declare.

scholarly journals CXCR4-SDF-1 Signaling in Nestin+ Mesenchymal Stem Cell Is Required for HSC Maintenance during Homeostasis and Regeneration after Irradiation

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 3883-3883 ◽  
Author(s):  
Pratibha Singh ◽  
Louis M. Pelus
Keyword(s):  
Stem Cells ◽  
Stromal Cell ◽  
Cxcr4 Expression ◽  
Wild Type ◽  
Post Transplantation ◽  
Hematopoietic Stem ◽  
Cxcr4 Signaling ◽  
Hematopoietic Recovery ◽  
Hematopoietic Reconstitution

Hematopoietic stem cells (HSC) reside in a complex microenvironment (niche) within the bone marrow (BM), where multiple populations of microenvironmental stromal cells regulate and finely tune their proliferation, differentiation and trafficking. Recent studies have shown that mesenchymal stem cells (MSC) are an essential component of the HSC niche. Intrinsic HSC CXCR4-SDF-1 signaling has been implicated in self-renewal and quiescence; however, the role of microenvironment CXCR4-SDF-1 signaling in supporting HSC function remains unclear. We previously demonstrated that microenvironmental stromal cell-derived CXCR4 is important for HSC recovery, as transplantation of wild-type HSC into CXCR4 deficient recipients showed reduced HSC engraftment. In this study, we now show that CXCR4-SDF-1 signaling in nestin+ MSC regulates HSC maintenance under normal homeostatic conditions and promotes hematopoietic regeneration after irradiation. Multivariate flow cytometry analysis of marrow stroma cells revealed that mouse BM MSCs identified as CD45-Ter119-CD31-Nestin+PDGFR+CD51+ express the CXCR4 receptor, which was confirmed by RT-PCR analysis. To investigate the role of MSC CXCR4 signaling in niche maintenance and support of HSC function, we utilized genetic mouse models, in which CXCR4 could be deleted in specific stromal cell types. Selective deletion of CXCR4 from nestin+ MSC in adult tamoxifen inducible nestin-cre CXCR4flox/flox mice resulted in reduced total MSC in BM (Control vs. Deleted: 647±128 vs. 209±51/femur, respectively, n=5, p<0.05), which was associated with a significant reduction in Lineage-Sca-1+c-Kit+ (LSK) cells (Control vs. Deleted: 18,033±439 vs. 4523±358/femur, respectively n=5, p<0.05). Selective CXCR4 deletion in nestin+ MSC also resulted in enhanced LSK cell egress to the peripheral circulation (Control vs. Deleted: 1022±106 vs. 2690±757/ml blood, respectively n=5, p<0.05), with no detectable difference in HSC cell cycle or apoptosis. However, the repopulation ability of HSC obtained from mice where CXCR4 was deleted in nestin+ MSC was reduced by >2 fold. In contrast, deletion of CXCR4 from osteoblasts using osteocalcin cre CXCR4flox/flox mice had no effect on HSC numbers in BM and blood.To investigate the role of nestin+ MSC CXCR4 signaling in BM niche reconstruction and hematopoietic recovery, we transplanted BM cells from wild-type mice into syngeneic wild-type or nestin+ MSC CXCR4 deleted recipients after lethal irradiation (950 rad) and analyzed HSC homing, niche recovery and hematopoietic reconstitution. Deletion of CXCR4 from nestin expressing MSC resulted in significantly reduced LSK cell homing at 16 hrs post transplantation (Control vs. Deleted: 8643±1371 vs. 3004±1044/ mouse, respectively, n=5, p<0.05). Robust apoptosis and senescence after total body irradiation was observed in nestin expressing MSCs lacking CXCR4 expression. At 15 days post-transplantation, chimeric mice with nestin+ MSC lacking CXCR4 expression displayed attenuated niche recovery and hematopoietic reconstitution compared to mice with wild-type stroma. In conclusion, our study suggests that CXCR4-SDF-1 signaling in nestin+ MSC is critical for the maintenance and retention of HSC in BM during homeostasis and promotes niche regeneration and hematopoietic recovery after transplantation. Furthermore, our data suggest the modulating CXCR4 signaling in the hematopoietic niche could be beneficial as a means to enhance HSC recovery following clinical hematopoietic transplantation or radiation/chemotherapy injury. Disclosures No relevant conflicts of interest to declare.

scholarly journals Hematopoietic recovery after transplantation is primarily derived from the stochastic contribution of hematopoietic stem cells

2021 ◽  
Author(s):  
Stefan Radtke ◽  
Mark Enstrom ◽  
Dnyanada P. Pande ◽  
Margaret L. Cui ◽  
Ravishankar Madhu ◽  
...  
Keyword(s):  
Clonal Diversity ◽  
Enhanced Sampling ◽  
Primate Model ◽  
Hematopoietic Stem ◽  
Neutrophil Recovery ◽  
Hematopoietic Recovery ◽  
Hematopoietic Reconstitution ◽  
Initial Recovery ◽  
Two Phases

Reconstitution after hematopoietic stem cell (HSC) transplantation is assumed to occur in two distinct phases: initial recovery mediated by short-term progenitors and long-term repopulation by multipotent HSCs which do not contribute to hematopoietic reconstitution during the first 6-9 months. We have previously reported the transplantation and exclusive engraftment of the HSC-enriched CD34+CD45RA-CD90+ phenotype in a nonhuman primate model. Here, we closely followed the clonal diversity and kinetics in these animals. Enhanced sampling and high density clonal tracking within the first 3 month revealed that multipotent HSCs actively contributed to the early phases of neutrophil recovery and became the dominant source for blood cells as early as 50 days after transplant. Longitudinal changes in clonal diversity supported a stochastic engraftment of HSCs with the majority of HSCs clones vanishing early during neutrophil recovery and a smaller fraction of HSC clones expanding into bigger pools to support long-term hematopoiesis. In contrast to the bi-phasic model, we propose that hematopoietic recovery after myeloablation and transplantation is primarily derived from HSCs in a stochastic manner rather than in two phases by independent cell populations.

scholarly journals The Notch Ligand Jagged-1 Represents a Novel Growth Factor of Human Hematopoietic Stem Cells

2000 ◽  
Vol 192 (9) ◽  
pp. 1365-1372 ◽  
Author(s):  
Frances N. Karanu ◽  
Barbara Murdoch ◽  
Lisa Gallacher ◽  
Dongmei M. Wu ◽  
Masahide Koremoto ◽  
...  
Keyword(s):  
Stem Cells ◽  
Growth Factor ◽  
Cultured Cells ◽  
Soluble Form ◽  
Human Stem Cells ◽  
Hematopoietic Stem ◽  
Notch Ligand ◽  
Immunodeficient Mice ◽  
Hematopoietic Reconstitution ◽  
Human Blood Cells

The Notch ligand, Jagged-1, plays an essential role in tissue formation during embryonic development of primitive organisms. However, little is known regarding the role of Jagged-1 in the regulation of tissue-specific stem cells or its function in humans. Here, we show that uncommitted human hematopoietic cells and cells that comprise the putative blood stem cell microenvironment express Jagged-1 and the Notch receptors. Addition of a soluble form of human Jagged-1 to cultures of purified primitive human blood cells had modest effects in augmenting cytokine-induced proliferation of progenitors. However, intravenous transplantation of cultured cells into immunodeficient mice revealed that human (h)Jagged-1 induces the survival and expansion of human stem cells capable of pluripotent repopulating capacity. Our findings demonstrate that hJagged-1 represents a novel growth factor of human stem cells, thereby providing an opportunity for the clinical utility of Notch ligands in the expansion of primitive cells capable of hematopoietic reconstitution.

scholarly journals HMGA2 promotes long-term engraftment and myeloerythroid differentiation of human hematopoietic stem and progenitor cells

2019 ◽  
Vol 3 (4) ◽  
pp. 681-691 ◽  
Author(s):  
Praveen Kumar ◽  
Dominik Beck ◽  
Roman Galeev ◽  
Julie A. I. Thoms ◽  
Mehrnaz Safaee Talkhoncheh ◽  
...  
Keyword(s):  
Cell Subset ◽  
High Mobility ◽  
Sequencing Analysis ◽  
Hematopoietic Stem ◽  
Growth And Survival ◽  
Hematopoietic Reconstitution ◽  
Cd34 Cells ◽  
Proliferation And Differentiation ◽  
Stem And Progenitor Cells

Abstract Identification of determinants of fate choices in hematopoietic stem cells (HSCs) is essential to improve the clinical use of HSCs and to enhance our understanding of the biology of normal and malignant hematopoiesis. Here, we show that high-mobility group AT hook 2 (HMGA2), a nonhistone chromosomal-binding protein, is highly and preferentially expressed in HSCs and in the most immature progenitor cell subset of fetal, neonatal, and adult human hematopoiesis. Knockdown of HMGA2 by short hairpin RNA impaired the long-term hematopoietic reconstitution of cord blood (CB)–derived CB CD34+ cells. Conversely, overexpression of HMGA2 in CB CD34+ cells led to overall enhanced reconstitution in serial transplantation assays accompanied by a skewing toward the myeloerythroid lineages. RNA-sequencing analysis showed that enforced HMGA2 expression in CD34+ cells induced gene-expression signatures associated with differentiation toward megakaryocyte-erythroid and myeloid lineages, as well as signatures associated with growth and survival, which at the protein level were coupled with strong activation of AKT. Taken together, our findings demonstrate a key role of HMGA2 in regulation of both proliferation and differentiation of human HSPCs.

scholarly journals Plerixafor alone for the mobilization and transplantation of HLA-matched sibling donor hematopoietic stem cells

2019 ◽  
Vol 3 (6) ◽  
pp. 875-883 ◽  
Author(s):  
Yi-Bin Chen ◽  
Jennifer Le-Rademacher ◽  
Ruta Brazauskas ◽  
Deidre M. Kiefer ◽  
Mehdi Hamadani ◽  
...  
Keyword(s):  
Hematological Malignancies ◽  
Hematopoietic Cell Transplantation ◽  
Multicenter Trial ◽  
Primary Objective ◽  
Hematopoietic Stem ◽  
Cd34 Cells ◽  
Matched Sibling ◽  
Multicenter Prospective Study ◽  
One Year ◽  
Stimulating Factor

Abstract Plerixafor, a direct antagonist of CXCR4/stromal-derived factor 1, can safely and rapidly mobilize allografts without the use of granulocyte colony-stimulating factor (G-CSF). We conducted a phase 2, multicenter, prospective study of plerixafor-mobilized HLA-identical sibling allografts for allogeneic hematopoietic cell transplantation in recipients with hematological malignancies. Donors (n = 64) were treated with subcutaneous plerixafor (240 µg/kg) and started leukapheresis (LP) 4 hours later. The primary objective was to determine the proportion of donors who were successfully mobilized: defined as collection of ≥2.0 × 106 CD34+ cells per kilogram recipient weight in ≤2 LP sessions. Recipients subsequently received reduced intensity (RIC; n = 33) or myeloablative (MAC; n = 30) conditioning. Sixty-three of 64 (98%) donors achieved the primary objective. The median CD34+ cell dose per kilogram recipient weight collected within 2 days was 4.7 (0.9-9.6). Plerixafor was well tolerated with only grade 1 or 2 drug-related adverse events noted. Bone pain was not observed. Plerixafor-mobilized grafts engrafted promptly. One-year progression-free and overall survivals were 53% (95% confidence interval [CI], 36% to 71%) and 63% (95% CI, 46% to 79%) for MAC and 64% (95% CI, 47% to 79%) and 70% (95% CI, 53% to 84%) for RIC recipients, respectively. Donor toxicity was reduced relative to G-CSF mobilized related donors. This is the first multicenter trial to demonstrate that, as an alternative to G-CSF, plerixafor rapidly and safely mobilizes sufficient numbers of CD34+ cells from matched sibling donors for HCT. Engraftment was prompt, and outcomes in recipients were encouraging. This trial was registered at clinicaltrials.gov as #NCT01696461.

Xenotransplantation of Primary CD34+CD38- Hematopoietic Stem Cells Results in an In Vivo Model of Idiopathic Myelofibrosis.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 666-666
Author(s):  
Noriyuki Saito ◽  
Fumihiko Ishikawa ◽  
Kazuya Shimoda ◽  
Shuro Yoshida ◽  
Yoriko Saito ◽  
...  
Keyword(s):  
Stem Cells ◽  
In Vivo Model ◽  
Idiopathic Myelofibrosis ◽  
Hematopoietic Stem ◽  
Hematopoietic Reconstitution ◽  
Cd34 Cells ◽  
Normal Human ◽  
Myelomonocytic Cells ◽  
Xenotransplantation Model

Abstract Idiopathic myelofibrosis (IMF) is characterized by clonal proliferation of abnormal myelomonocytic cells and megakaryocytes. These abnormal cells secrete various cytokines resulting in reactive fibrosis and increased collagen content in the bone marrow (BM), and lead to extramedullary hematopoiesis and the appearance of CD34+ cells in the peripheral blood (PB). Although IMF is thought to originate at the level of hematopoietic stem cell (HSC), this has not been demonstrated directly in primary human IMF. To demonstrate the involvement of HSCs in the pathogenesis of IMF and to establish an in vivo model of IMF, we used the newborn NOD/SCID/IL2rg-null xenotransplantation model. We purified PB CD34+ cells from six IMF patients, transplanted 1–10 x10e4 cells intravenously into newborn NOD/SCID/IL2rg-null recipients and analyzed PB and BM human CD45+ hematopoietic cell chimerism, degree of suppression of murine hematopoiesis, presence of hallmark BM fibrosis and plasma TGF-b1 levels in the recipients at 6 months post-transplantation. Primary IMF PB CD34+ cells from five out of six patients engrafted in twelve out of twelve recipients. BM of all engrafted recipients demonstrated fibrotic changes associated with increased proliferation of murine fibroblasts, the presence of human megakaryocytes and elevated plasma TGF-b1 levels, recapitulating the clinical features of IMF. Three distinct patterns of human hematopoietic reconstitution were observed among the engrafted recipients: Predominantly malignant myelomonocytic engraftment in the PB and BM (n=4), Reconstitution of both normal human hematopoiesis (with mature B and T cells, myeloid cells and platelets) and malignant myelomonocytic cells (n=6) and Development of acute leukemia (n=2). Fibrotic change was seen even in the BM of recipients that showed normal human hematopoietic reconstitution, showing that in IMF, there is co-existence of both normal and malignant hematopoietic stem/progenitor cells in the PB CD34+ fraction. Furthermore, when 5–10 x 10e3 sorted PB CD34+CD38– cells from three patients were transplanted into six newborn NOD/SCID/IL2rg-null recipients, reconstitution with human myelomonocytic cells associated with BM fibrosis was demonstrated in all recipients, with compatible level of PB and BM chimerism with those transplanted with PB CD34+ cells. These findings demonstrate that the IMF-initiating cells are contained within the HSC fraction. The newborn NOD/SCID/IL2rg-null xenotransplantation model provides an in vivo model of primary human IMF that may lead to better understanding of the mechanisms of IMF pathogenesis including the identification of IMF stem cells and may be useful for development of novel therapeutic agents for IMF.

The Dose of CD34+ Cells Negatively Impacts the Outcome of Patients Treated with Genoidentical Allo SCT Prepared with a Reduced Intensity Conditioning (RIC).

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1086-1086
Author(s):  
Didier Blaise ◽  
Laure Farnaut ◽  
Catherine Faucher ◽  
Sabine Furst ◽  
Jean El Cheikh ◽  
...  
Keyword(s):  
Hematopoietic Cell Transplantation ◽  
Cox Model ◽  
Disease Status ◽  
Probability Estimates ◽  
Cd34 Cells ◽  
Comorbidity Index ◽  
And Function ◽  
Donor Source

Abstract While RIC are presently commonly used, most of the reports suffer from insufficiencies limiting knowledge acquisition: small populations, short follow-up, heterogeneities in the RIC intensity or the donor source. We report here 100 patients with hematological malignancies (HM) treated with geno-identical SCT after the same RIC in a single center from 2000 until 2006. All patients received oral busulfan (8mg/m2), thymoglobulin (2.5 mg/kg) and Fludarabine (150 to 180 mg/m²) (FBT conditioning). All grafts were PBSC from a match sibling donor. All grafts were monitored for CD34, CD3, CD4, CD8, CD19 and CD56+ cells (notably CD34= 5.6 (1.5–22) × 10e6/kg; CD3= 317 (112–887) × 10e6/kg). Median age was 50 (18–64). Hematopoietic cell transplantation comorbidity index (HCT-CI) was 0, 1–2 and > 2 in 31, 39 and 23 of the 93 evaluable patients. All patients received post-graft CSA. Diagnoses included acute leukemia (39%), Myeloid (16%) or Lymphoid (45%) malignancies. 53, 14 and 33 pts were in CR, progression or stable disease. Minimal and median follow-up are respectively 6 and 34 months. All but one engrafted reaching full lymphoid donor chimerism prior to day 100 in 85% of the cases. 55 pts presented aGVHD (G1: 12; G2: 22; G3: 12; G4: 9) for a cumulative incidence (CI) of G2–4 aGVHD of 43% (33–53); 91 patients were evaluable for cGVHD with a 79% (71–87) CI (Lim= 20%; Ext: 80%). In a multivariate analysis cGVHD occurrence could be predicted by 2 independent factors: lower dose of CD34 (but no impact of CD3, CD4, CD8, CD19 or CD56) (odd ratio (OR): 0.79 (0.69–0.90)) and grade 2–4 aGVHD (OR: 1.16 (1.01–1.32)). TRM CI was respectively 5%, 14% and 19% (11–27) at 3, 12 months and overall. TRM was strongly associated with aGVHD occurrence (TRM CI: grade 2–4 aGVHD: 37% (23–51); grade 0–1 aGVHD: 7% (0–14): p<.01). Relapse CI was 15% (8–22) at a median of 169 days (30–769). Disease control was significatively associated with cGVHD occurrence (Relapse CI: cGVHD: 10% (3–17); no cGVHD: 42% (14–70): p=.01). 5 year overall survival (OS) and LFS probability estimates are 62% (50–72) and 60% (48–72) with a plateau starting after 3.5 years. Outcome was similar for the patients above or under 50 and for the different diagnoses. HCT-CI did not show any influence on outcome. In a Cox model analysis, LFS was independently affected by only 2 pre-transplant variables: disease status (CR vs. no CR) (OR=0.45; p=.022) and the dose of infused CD34+ cells (> or < 5.6 × 10e6 CD34+ cells) (OR: 2.04; p=.039). In accordance with above, patients with higher CD34+ cells presented less cGVHD (cGVHD CI: CD34 < 5.6: 98% (94–100); CD34 > 5.6: 76% (64–88) ; p<0.01). These data confirm that FBT RIC, combining myeloablation (Busulfan) and limited Thymoglobulin, is efficient in a wide population in term of age and diagnosis conducting to sustained long term OS and EFS with limited TRM. They suggest also that after RIC, graft composition and function probably in relation with G-CSF mobilization might impact allogeneic effect and invite revisiting this subject in this context.

Kinetics of Engraftment and Graft Versus Host Disease After Cotransplantation of Ex Vivo Expanded and Unexpanded Cord Blood Units In Immunodeficient Mice.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3722-3722
Author(s):  
Li Ming Ong ◽  
Xiubo Fan ◽  
Pak Yan Chu ◽  
Florence Gay ◽  
Justina Ang ◽  
...  
Keyword(s):  
Cord Blood ◽  
Conflicts Of Interest ◽  
Ex Vivo ◽  
Ex Vivo Expansion ◽  
Hematopoietic Stem ◽  
Immunodeficient Mice ◽  
Insulin Growth Factor ◽  
Nsg Mice ◽  
Nonobese Diabetic ◽  
Cd34 Cells

Abstract Abstract 3722 Ex vivo expansion of cord blood (CB) hematopoietic stem cells (HSCs) and cotransplantation of two CB units can enhance applicability of CB transplants to adult patients. This is the first study on cotransplantation of ex vivo expanded and unexpanded human CB units in immunodeficient mice, simulating conditions for ex vivo CB expansion clinical trials. CB units were cultured in serum-free medium supplemented with Stem Cell Factor, Flt-3 ligand, Thrombopoietin and Insulin Growth Factor Binding Protein-2 with mesenchymal stromal co-culture. Cotransplantation of unexpanded and expanded CB cells was achieved by tail vein injection into forty-five sublethally irradiated nonobese diabetic SCID-IL2γ−/− (NSG) mice. Submandibular bleeding was performed monthly and mice were sacrificed 4 months following transplantation to analyze for human hematopoietic engraftment. CB expansion yielded 40-fold expansion of CD34+ cells and 18-fold expansion of HSCs based on limiting dilution analysis of NSG engraftment. Mice receiving expanded grafts had 4.30% human cell repopulation, compared to 0.92% in mice receiving only unexpanded grafts at equivalent starting cell doses (p = 0.07). Ex vivo expanded grafts with lower initiating cell doses also had equivalent engraftment to unexpanded grafts with higher cell dose (8.0% vs 7.9%, p= 0.93). However, the unexpanded graft, richer in T-cells, predominated in final donor chimerism. Ex vivo expansion resulted in enhanced CB engraftment at equivalent starting cell doses, even though the unexpanded graft predominated in long-term hematopoiesis. The expanded graft with increased stem/progenitor cells enhanced initial engraftment despite eventual rejection by the unexpanded graft. Disclosures: No relevant conflicts of interest to declare.

Proof of Benefit in Multiple-Cord Blood Transplantation Evidenced By Early Hematopoietic Reconstitution

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 3071-3071
Author(s):  
Takashi Ishida ◽  
Satoshi Takahashi ◽  
Chen-Yi Lai ◽  
Masaaki Higashihara ◽  
Hiromitsu Nakauchi ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
Human Cell ◽  
Single Unit ◽  
Bone Marrow Cells ◽  
Donor Chimerism ◽  
Single Donor ◽  
Hematopoietic Reconstitution ◽  
Cd34 Cells ◽  
New Strategy

Abstract Introduction: Umbilical cord blood (UCB) serves as a suitable donor source in hematopoietic cell transplantation. However, the initial time to engraftment in UCB transplantation (UCBT) is delayed because of its insufficient graft cell numbers, which often limits its use in UCBT. According to the report by Japanese UCB bank, the majority of UCB units remain unused clinically because of their low graft cell doses. Therefore, we sought to develop a new strategy to improve outcomes of UCBT by using multiple (more than 3) units to augment graft cell doses. We reported at the ASH meeting 2014 that an early hematopoietic recovery in lethally irradiated mice was observed by combining hematopoietic stem/progenitor cells (HSC/HSPCs) derived from multiple allogeneic mouse strains. Herein to establish a clinically relevant procedure, we provide a proof of benefit of this approach using human UCB cells in xenotransplantation models. Methods: We initially subjected a single UCB (HLA-A2 negative) to CliniMACS Prodigy®(Miltenyi Biotec, Bergisch Gladbach, Germany) to acquire mononuclear cells (MNCs). Obtained MNCs were further processed for isolation of either CD34+ cells or CD3+ T cells using microbeads techniques (Figure 1, Unit A). We next combined 3 frozen UCB units (Units B-D, all HLA-A2 positive) into one bag after thawing and then applied them to CliniMACS Prodigy®. Mixed CD34+ cells were purified from obtained MNCs. These test CD34+ cells were transplanted into sublethally-irradiated immunodeficient mice. Mice in the first group received single unit CD34+ cells alone (Unit A, G1 in Figure 2, 3.2 x 104 cells). The additive effect on human cell engraftment was tested in mixed CD34+ cells by transplanting them (Units B-D, G2, 9.6 x 104 cells) together with the single unit CD34+ cells (Unit A, 3.2 x 104 cells). Another cohort of mice (G3) received Unit A CD34+ cells at a dose 4 times more than that in G1 (1.28 x 105 cells). We also tested whether the inclusion of CD3+ T cells (2 x 105 cells) derived from one UCB (Unit A) favored formation of single donor chimerism. Detailed donor cell chimerism was determined in bone marrow over time by flow cytometry analysis, using HLA subtypes as indicators of donor origins. Results: As formerly reported, in a multiple-donor transplantation model using mouse bone marrow cells, combined multiple units of allogeneic HSC/HSPCs were shown to protect lethally-irradiated recipient mice, and to accelerate the recovery of granulocytes, hemoglobin and platelets when infused additionally with a single graft that was composed of whole bone marrow cells. Single donor chimerism was achieved long-term in these mice. Experiments using sorted population of a graft demonstrated that an appropriate dose of T cells should be contained within the primary unit to achieve single donor chimerism through graft versus graft reactions. We then tested whether our strategy was applicable to human UCB cells in xeno-transplantation models. At the first attempt, it was extremely difficult to extract viable CD34+ cells manually from frozen UCB units. To overcome this problem, we employed CliniMACS Prodigy®. Irrespective of HLA type disparities, we eventually succeeded in extracting viable MNCs from multiple UCB units at one time by using CliniMACS Prodigy®. Purified cells were obtained from them using microbeads techniques, with %CD34 and %CD3 of the final test cells being more than 98 % and 96 %, respectively. Up to 9 UCB units were applicable to CliniMACS Prodigy® together at once. As shown in Figure 2, combined multiple UCB units exhibited robust hematopoietic reconstitution. The human cell chimerism in G2 (mixed units) was as high as that observed in G3 (single unit with the same dose). As evidenced by sequential chimerism analysis, inclusion of T cells in one graft achieved single donor chimerism following transient mixed donor chimerism. Conclusions: Our study uncovered that combined multiple UCB units of allogeneic HSC/HSPCs were capable of accelerating hematopoietic reconstitution when manipulated appropriately and added to an otherwise "insufficient dose" setting of transplantation both for mice and human cases. Development of a new strategy of multiple UCB transplantation will open a door for wider application of UCBT. Disclosures No relevant conflicts of interest to declare.

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