Human Amniotic Fluid Stem Cells Have Hematopoietic Potential In Vivo

Abstract Introduction Human hematopoietic stem cell transplantation (HSCT) has been used to treat a range of hematological and immunological disorders. As a result, the demand for hematopoietic stem cells (HSC) in clinical applications is increasing. Amniotic fluid stem cells (AFSC) serve as a potential alternative cell source for therapy. Amniotic fluid can be derived by amniocentesis or therapeutic amniodrainage. AFSC are multi-potent, have low risk of tumorigenicity, can be expanded and do not have legal or ethical limitations. The significant hematopoietic activity of murine AFSC led us to explore the potential of human AFSC (CD117/c-Kit+) towards hematopoietic differentiation and to reconstitution in vivo. Methods Human AFSC (2nd and 3rd trimester) and cord blood HSC (CB-HSC; control) were selected for CD117 and CD34 respectively using a MoFlo XDP sorter. Sorted cells (104 in 200μl PBS) were injected intravenously into sub-lethally irradiated NOD-SCID/IL2rγnull (NSG) mice (n=6/group). Hematopoietic engraftment of human cells (% of human CD45+ within total CD45+) and multi-lineage reconstitution (myeloid: CD13, CD14, CD15 and lymphoid: CD3, CD4 and CD8) were assessed at 16 weeks in blood, bone marrow (BM) and spleen by flow cytometry. For subsequent secondary transplants, BM mononuclear cells (MNC) derived from BM harvested from primary recipients of mice were intravenously injected into secondary recipients (1.5x107 MNC in 200μl PBS). Hematopoietic engraftment was assessed at 16 weeks post-transplantation (n=6/group). For further analysis of human donor cell engraftment, Q-PCR was performed on spleen samples harvested from primary and secondary recipients using oligonucleotide primers specific for human ALU repeat sequences; Immunohistochemistry was carried out using anti-human CD45 antibody and detected with a commercially available kit (Dako EnVision Plus, Dako). Results are expressed as mean±SEM, and statistical analysis was performed using 1-way ANOVA with Bonferroni post-hoc tests. Results Human AFSC engrafted the hematopoietic system of NSG mice at levels similar to the ones achieved with CB-HSC (blood: AFSC 7.5±1.3% vs. CB-HSC 6.1±2.2%, p=0.6; BM: AFSC 46.3±7.9% vs. CB-HSC 38.3±8.2%, p=0.6; spleen: AFSC 39.6±9.3% vs. CB-HSC 34.7±10.5%, p=0.7). Similarly, at 16 weeks following secondary transplantation, human donor cell engraftment was comparable between groups in blood (AFSC 11.5 ± 3.9% vs. CB-HSC 16.9 ± 3.9%, p=0.3) and other hematopoietic tissues. Q-PCR and immunohistochemistry confirmed donor cell engraftment in AFSC and CB-HSC groups. Importantly, there were no differences between groups in multi-lineage differentiation at 16 weeks post primary and secondary transplantation. Conclusion Human CD117/c-Kit+ AFSC have functional, multi-lineage hematopoietic potential that is similar to the current "gold-standard" stem cell source for hematopoietic transplantation. The ease of isolation during early gestation, as well as their gene-engineering and expansion potential make human AFSC a novel autologous fetal cell source for pre- and post-natal therapy of inherited hematological disorders. Disclosures No relevant conflicts of interest to declare.

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Effects of Sickle Cell Disease on Hematopoietic Stem Cell Function and the Bone Marrow Microenvironment.

Blood ◽

10.1182/blood.v108.11.1227.1227 ◽

2006 ◽

Vol 108 (11) ◽

pp. 1227-1227

Author(s):

Elisabeth H. Javazon ◽

Leslie S. Kean ◽

Jennifer Perry ◽

Jessica Butler ◽

David R. Archer

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Stem Cell ◽

Sickle Cell Disease ◽

Sickle Cell ◽

Cell Function ◽

Donor Cell ◽

Cell Disease ◽

Hematopoietic Stem ◽

Cell Engraftment

Abstract Gene therapy and stem cell transplantation are attractive potential therapies for sickle cell disease (SCD). Previous studies have shown that the sickle environment is highly enriched for reactive oxygen species (ROS), but have not addressed whether or not the increased ROS may alter the bone marrow (BM) microenvironment or affect stem cell function. Using the Berkeley sickle mouse model, we examined the effects of sickle cell disease on hematopoietic stem cell function and the bone marrow microenvironment. We transplanted C57BL/6 (control) BM into C57BL/6 and homozygous sickle mice. Recipients received 2 × 106 BM cells and a conditioning regimen consisting of busulfan, anti-asialo GM1, and co-stimulation blockade (anti-CD40L and CTLA4-Ig). Following transplantation, sickle mice demonstrated increased donor cell engraftment in the peripheral blood compared to normal mice (58.3% vs. 33.1%, respectively). Similarly, BMT in a fully allogeneic system also resulted in enhanced engraftment in sickle recipients. Next we analyzed whether or not engraftment defects exist within the BM stem cell population of sickle mice. In vitro colony forming assays showed a significant decrease in progenitor colony formation in sickle compared to control BM. By flow cytometry, we determined that there was a significant decrease in the KSL (c-Kit+, Sca-1+, Lineage−) progenitor population within the BM of sickle mice. Cell cycle analysis of the KSL population demonstrated that significantly fewer sickle KSL cells were in G0 phase compared to control, suggesting that there are fewer quiescent stem cells in the BM of sickle mice. To assess the potential role of ROS and glutathione depletion in sickle mice, we tested the engraftment efficiency of KSL cells from untreated and n-acetyl-cysteine (NAC) treated control, hemizygous sickle (hemi), and sickle mice in a competitive repopulation experiment. Peripheral chimerism showed an engraftment defect from both hemizygous and homozygous sickle mice such that control KSL cells engrafted > hemi > sickle at a ratio of 1 : 0.4 : 0.25. Treatment with NAC for four months prior to transplantation partially restored KSL engraftment (control : hemi : sickle; 1 : 0.97 : 0.56 ). We have demonstrated that congenic and allogeneic BMT into sickle mice result in increased donor cell engraftment in the sickle recipients. Both the decreased number of KSL cells and the decreased percentage of quiescent KSL cells in the sickle mice indicate that more stem cells in the transgenic sickle mouse model are mobilized from the BM environment. The engraftment defect of sickle KSL cells that was partially ameliorated by NAC treatment suggests that an altered redox environment in sickle mice may contribute to the engraftment deficiencies that we observed.

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Functional Genomics and Computational Approaches Identify Novel Small Molecules Targeting Quiescent Leukemia Stem Cells

Blood ◽

10.1182/blood.v126.23.1391.1391 ◽

2015 ◽

Vol 126 (23) ◽

pp. 1391-1391

Author(s):

Costakis Frangou ◽

Jason Den Haese ◽

Jordan Warunek ◽

Scott Portwood ◽

Norma J Nowak ◽

...

Keyword(s):

Stem Cells ◽

Drug Resistance ◽

Signaling Pathways ◽

Research Funding ◽

Cell Tracking ◽

Leukemia Stem Cells ◽

Self Renewal ◽

Hematopoietic Stem ◽

Nsg Mice

Abstract Chemotherapy or targeted cancer therapies have greatly improved the treatment outcome of patients with leukemia; however, many will ultimately die because of disease relapse and development of drug resistance. Leukemias are cancers of the blood cells that result from alteration of the normal physiological constraints that regulate hematopoietic stem cells (HSCs). General characteristics of leukemia stem cells (LSCs) such as self-renewal, self-protection and proliferative quiescence represent inherent mechanisms that at least partially explain drug resistance and recurrence in post-therapy leukemia patients. Acute myeloid leukemia (AML) is a heterogeneous disease, both biologically and clinically, in which a number of distinct genetic abnormalities have been described. Several recent studies suggest that this heterogeneity extends to LSCs and can vary between patient subgroups, and even within individual patients. Moreover, the complexity of AML is further complicated by the existence of functionally diverse leukemic and preleukemic clones. Accordingly, the hierarchical organization of AML suggests that this may be relevant to current therapies that primarily target proliferating progenitors/blast cells, which lack self-renewal capacity, and not LSCs. In the current study, we rationalized that understanding how LSCs differ from normal HSCs at the molecular level, is an essential first step towards developing novel targeted therapies and achieving permanent disease remission. Despite the identification of novel LSC-specific markers, there is considerable heterogeneity in expression of these markers amongst AML patients. However, in addition to marker-enrichment strategies, LSCs can be identified by virtue of their quiescent and slow-cycling properties. For example, label-retaining cells can be isolated and used in functional assays but significant technical limitations impede broad utility of this approach. To this end, we describe the development and use of novel multi-fluorescent protein markers and DNA bar codes integrated into the cellular genomes by lentivirus, as single-cell tracking devices for monitoring LSCs in vivo. We demonstrate how LSCs can transition between a "proliferation phase" and a "quiescence phase" in vivo. Furthermore, using high-throughput quantitative transcriptome sequencing (Q-RNA-Seq) and RNAi genetic perturbation's focusing on well-defined self-renewal signaling pathways, we develop a differential network-based model to identify LSC-specific genes and subsequently prioritize/rank candidates as potential drug targets. In the current study, we identify several molecular targets deregulated in quiescent versus proliferating LSCs and a mutual set of signaling pathways that facilitate leukemic transformation downstream of diverse initiating mutations/lesions. Remarkably, both quiescent and dividing LSCs but not HSCs, were 'addicted' to SSRP1 - an essential component of the ubiquitous FACT chromatin remodeling complex. Two orally available quinacrine-related DNA-intercalating compounds inhibiting function of FACT (CBL0100 and CBL0175, respectively) suppressed LSC proliferation in vitro and in vivo, as demonstrated by production of leukemic clonogenic cells (CFU) and long-term engraftment of immunodeficient NSG mice, by simultaneous inhibition of NF-kB (stimulated and basal forms) and activation of p53. Furthermore, in a secondary transplantation experiment, leukemic cells obtained from CBL0175 treated mice (primary) failed to engraft into secondary NSG mice in a serial transplantation model by selectively targeting the LSC compartment. Collectively, we present a novel network-based polypharmacology approach that provides unique opportunities to preferentially ablate LSCs (quiescent and dividing types), with potentially profound clinical implications. Disclosures Frangou: Cellecta: Employment. Portwood:ImmunoGen: Research Funding. Wang:ImmunoGen: Research Funding.

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Amniotic Fluid Stem Cells Rescue Both in Vitro and in Vivo Growth, Innervation, and Motility in Nitrofen-Exposed Hypoplastic Rat Lungs through Paracrine Effects

Cell Transplantation ◽

10.3727/096368912x657756 ◽

2013 ◽

Vol 22 (9) ◽

pp. 1683-1694 ◽

Cited By ~ 44

Author(s):

F. Pederiva ◽

M. Ghionzoli ◽

A. Pierro ◽

P. De Coppi ◽

J. A. Tovar

Keyword(s):

Stem Cells ◽

Amniotic Fluid ◽

Paracrine Effects ◽

Amniotic Fluid Stem Cells ◽

Rat Lungs ◽

In Vivo Growth

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Hematopoietic Stem Cell Engraftment in Bone Marrow Is Dependent upon Gsα.

Blood ◽

10.1182/blood.v108.11.857.857 ◽

2006 ◽

Vol 108 (11) ◽

pp. 857-857

Author(s):

Gregor B. Adams ◽

Ian R. Alley ◽

Karissa T. Chabner ◽

Ung-il Chung ◽

Emily S. Marsters ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Stem Cell ◽

Bone Marrow Cells ◽

Fetal Liver ◽

Conditional Knockout ◽

Wild Type ◽

Hematopoietic Stem ◽

Cell Engraftment

Abstract During development, hematopoietic stem cells (HSCs) translocate from the fetal liver to the bone marrow, which remains the site of hematopoiesis throughout adulthood. In the bone marrow the HSCs are located at the endosteal surface, where the osteoblasts are a key component of the stem cell niche. The exogenous signals that specifically direct HSCs to the bone marrow have been thought to include stimulation of the chemokine receptor CXCR4 by its cognate ligand stromal derived factor-1α (SDF-1α or CXCL12). However, experiments in which CXCR4−/− fetal liver hematopoietic cells were transplanted into wild-type hosts demonstrated efficient engraftment of the HSCs in the bone marrow. In addition, treatment of HSCs with inhibitors of Gαi-coupled signaling, which blocks transmigration towards SDF-1αin vitro, does not affect bone marrow homing and engraftment in vivo. Therefore, we examined whether Gsα-coupled mechanisms play a key role in the engraftment of the HSCs in the bone marrow environment. Utilizing an inducible-conditional knockout of Gsα, we found that deletion of the gene in hematopoietic bone marrow cells did not affect their ability to perform in the in vitro primitive CFU-C or LTC-IC assay systems. However, Gsα−/− cells were unable to establish effective hematopoiesis in the bone marrow microenvironment in vivo in a competitive repopulation assay (41.1% contribution from wild-type cells versus 1.4% from knockout cells). These effects were not due to an inability of the cells to function in the bone marrow in vivo as deletion of Gsα following establishment of hematopoiesis had no effects on the HSCs. Examining the ability of the HSCs to home to the bone marrow, though, demonstrated that deletion of Gsα resulted in a marked impairment of the ability of the stem cells to localize to the marrow space (approximately 9-fold reduction in the level of primitive cell homing). Furthermore, treatment of BM MNCs with an activator of Gsα augmented the cells homing and thus engraftment potential. These studies demonstrate that Gsα is critical to the localization of HSCs to the bone marrow. Which receptors utilize this pathway in this context remains unknown. However, Gsα represents a previously unrecognized signaling pathway for homing and engraftment of HSCs to bone marrow. Pharmacologic activation of Gsα in HSC ex vivo prior to transplantation offers a potential method for enhancing stem cell engraftment efficiency.

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ZFN-Driven Gene Editing Prevents HLA-A Expression On Hematopoietic Stem Cells -Improving The Chance Of Finding An HLA-Matched Donor

Blood ◽

10.1182/blood.v122.21.1655.1655 ◽

2013 ◽

Vol 122 (21) ◽

pp. 1655-1655

Author(s):

Hiroki Torikai ◽

Tiejuan Mi ◽

Sonny O Ang ◽

Loren Gragert ◽

Martin Maiers ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

T Cells ◽

Hematopoietic Stem Cells ◽

Donor Pool ◽

Hematopoietic Stem ◽

Nsg Mice ◽

Immune Mediated ◽

Matched Donor

Abstract Hematopoietic stem cells (HSCs) are administered (i) to restore hematopoiesis and immunity in the course of hematopoietic stem-cell transplantation (HSCT), (ii) as a replacement for inherited blood disorders and bone marrow failure, (iii) to regenerate cells of alternative lineages for restorative medicine, and (iv) as a source for generating specific hematopoietic cells (e.g., T cells, NK cells, and dendritic cells). However, the widespread application of allogeneic HSCs for humans is hampered by their immune-mediated destruction by host T cells recognizing mismatched HLA or by HLA-specific antibodies. Despite pre-banking umbilical cord blood (UCB) units and access to adult donors through the National Marrow Donor Program (NMDP), finding a suitable HLA-matched product is challenging for many recipients, especially those from ethnic minorities who are under-represented in the donor pool. The available donor pool would be markedly increased if donor HSCs were edited to eliminate expression of the HLA-A locus. Indeed, modeling from NMDP shows that the chance of an African American recipient finding a HLA-matched donor increases from 18% to 73% when matched for HLA-B, C and DR, instead of HLA-A, B, C and DR. We have previously shown that engineered zinc finger nucleases (ZFNs) can disrupt HLA-A expression in genetically edited T cells (Blood 2013). To extend this proof-of-concept to HSCs, we sought to disrupt HLA-A expression by introducing ZFNs targeting this locus. CD34+lineageneg HSCs (99% purity) were isolated using paramagnetic beads from UCB. Electro-transfer of in vitro transcribed mRNA encoding the HLA-A-specific ZFN generated 30% HLA-Aneg HSCs after one week ex vivo culture with defined cytokines (FLT3-L, SCF, TPO, and IL-6) and an aryl hydrocarbon receptor antagonist (stem reginin-1, SR-1). As expected, SR1 treatment maintained greater numbers of CD34+ cells (also CD34posCD38neg) in culture compared to controls. DNA sequence analysis revealed that HLA-Aneg HSCs encode the expected nucleotide changes at the ZFN target site. An in vivo engraftment assay, using NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) mice, demonstrated that HLA-Aneg HSCs maintain the capability of engraftment and differentiation into HLA-Aneg hematopoietic cells (Figure). Thus, disruption of HLA-A expression in HSCs provides an appealing approach to increasing the chances for of finding HLA-matched donors and may broaden the clinical application of allogeneic HSCT. Furthermore, the ability to genetically edit HSCs has implications for (i) preventing immune-mediated recognition of HLA-disparate HSC and (ii) preventing immune mediated recognition of self-antigens. Engraftment of HLA-A2neg HSCs was evaluated in vivo. Data shown are flow-cytometry analysis of bone marrow obtained from NSG mice 16 weeks after HSC injection. HSC engraftment and HLA-A2 expression in NSG mice injected with un-modified HSCs (left panel) and HSCs treated with the HLA-A specific ZFNs (right panel) are shown. Data are gated on human CD45 positive cells. Figure Engraftment of HSCs modified by the HLA-A specific ZFNs in NSG mice. Figure. Engraftment of HSCs modified by the HLA-A specific ZFNs in NSG mice. Disclosures: Reik: Sangamo BioSciences: Employment. Holmes:Sangamo BioSciences: Employment. Gregory:Sangamo BioSciences: Employment.

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Heterogeneity Of Leukemic Stem Cell Capacity Of BCR-ABL+ Long-Term Hematopoietic Stem cells In CML Is Associated With Variability In MPL Expression

Blood ◽

10.1182/blood.v122.21.516.516 ◽

2013 ◽

Vol 122 (21) ◽

pp. 516-516

Author(s):

Bin Zhang ◽

Yin Wei Ho ◽

Wei Tong ◽

Ling Li ◽

Ravi Bhatia

Keyword(s):

Stem Cells ◽

Stem Cell ◽

Cell Growth ◽

Hematopoietic Stem Cells ◽

Myelogenous Leukemia ◽

Hematopoietic Stem ◽

Nsg Mice ◽

Donor Cells

Abstract In chronic myelogenous leukemia (CML), in vivo long-term repopulating and leukemia stem cell (LSC) capacity is restricted to a small population of BCR-ABL+ long-term hematopoietic stem cells (LTHSC). Using an inducible transgenic SCL-tTA/BCR-ABL mouse model of CML, we have shown that leukemic cells with long-term repopulating and leukemia-initiating capacity have the Lin-Sca-1+Kit+Flt3-CD150+CD48- phenotype, also characteristic of normal LTHSC. Limiting dilution transplantation studies show that frequency of cells with LTHSC phenotype with long-term engraftment capacity (1:6) is considerably higher than those with leukemia-initiating capacity (1:80) suggesting that only some LTHSC may have LSC capacity (Cancer Cell 21:577, 2012). To further evaluate the basis for heterogeneity in LSC potential of BCR-ABL+ LTHSC, SCL-tTA/BCR-ABL mice were crossed with GFP expressing mice to allow tracking of donor cells, and a cohort of mice were transplanted with limiting numbers of GFP+LTHSC (200 per mouse) and followed for engraftment of GFP+ cells and development of CML (WBC>10,000/ul). Only 11 of 20 mice developed CML, whereas 9 mice showed long term engraftment without development of CML. GFP+ LTHSC selected from primary recipients were transplanted into secondary recipients (200 per mouse). Seven of 17 mice receiving cells from mice with CML also developed CML after the second transplant, whereas none of the mice receiving cells from non-CML mice developed CML, suggesting the distinction between leukemogenic versus non-leukemogenic LTHSC was maintained after transplantation. LTHSC isolated from primary recipients were also analyzed for expression of several HSC-regulatory genes by multiplex Q-PCR using the Fluidigm system. On hierarchical clustering, LTHSC from mice developing CML clustered separately from LTHSC from mice without CML. Amongst cell surface expressed genes, expression of the thrombopoietin (TPO) receptor MPL (p=0.006) and CD47 (p=0.006) was significantly increased in LTHSC from mice developing CML. We did not see significant differences in BCR-ABL expression in LTHSC from mice with or without CML. We further analyzed the relationship of MPL expression with CML LTHSC function. CML LTHSC (n=6) expressing high levels of MPL (MPLhi, top 10% based on MPL expression) showed significantly increased cell growth (p<0.0001) and CFC potential (p=0.0007) when cultured with TPO (10ng/ml) compared to LTHSC expressing low levels of MPL (MPLlo, lowest 10% based on MPL expression), as well as significantly increased cell growth (p=0.005) and CFC (p=0.03) compared to normal MPLhi LTHSC. Following transplantation, MPLhi LTHSC (200 per mouse) generated significantly higher short-term (4 wks, p=0.008) and long-term (16 wks, p=0.003) engraftment of donor cells compared to MPLlo LTHSC. Seven of 16 mice receiving MPLhi LTHSC developed CML compared to only 1 out of 17 mice receiving MPLlo LTHSC. We next evaluated heterogeneity of MPL expression in LTHSC (CD34+CD38-CD90+ cells) from CML patients and normal subjects. As was seen in murine studies, human CML MPLhi LTHSC cultured with TPO (10ng/ml) showed increased cell growth (p<0.0001) and CFC frequency (p=0.02) compared to CML MPLlo LTHSC, and significantly increased cell growth (p<0.0001) and CFC generation (p=0.02) compared to normal MPLhi LTHSC. Both baseline and TPO stimulated p-Stat3/5 levels were significantly higher in human CML MPLhi LTHSC compared with MPLlo LTHSC (p<0.0001), and in CML compared to normal MPLhi LTHSC. Interestingly p-Stat5 response peaked at 1 hour in CML LTHSC compared to 20 minutes in normal LTHSC, further indicating alterations in MPL signaling in CML LTHSC. Transplantation of CML MPLhi LTHSC (3x104 cells/mouse) into NSG mice resulted in higher engraftment of human myeloid cells in BM at both 4 and 16 weeks (p<0.05) compared with MPLlo LTHSC. Normal MPLhi LTHSC also showed higher engraftment in NSG mice at 4 and 16 weeks compared with MPLlo cells. Our studies indicate that CML LTHSC represent a heterogeneous population with varying LSC capacity. Heterogeneity in LSC capacity is associated with variability in expression of MPL. Higher levels of MPL expression in CML LTHSC are associated with significantly increased Stat3/5 signaling, in vitro and in vivo growth, and LSC capacity. These results identify MPL as a key regulator of LSC potential of BCR-ABL+ LTHSC and a potential target for LSC-directed therapeutics. Disclosures: No relevant conflicts of interest to declare.

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Hematopoietic Engraftment of Amniotic Fluid Stem Cells Following in Utero Transplantation

Blood ◽

10.1182/blood.v124.21.3809.3809 ◽

2014 ◽

Vol 124 (21) ◽

pp. 3809-3809 ◽

Cited By ~ 1

Author(s):

Stavros P Loukogeorgakis ◽

Enrica Bertin ◽

Panicos Shangaris ◽

Chiara Franzin ◽

Aimee G Kim ◽

...

Keyword(s):

Stem Cells ◽

Flow Cytometry ◽

Amniotic Fluid ◽

Donor Cell ◽

In Utero ◽

Prenatal Treatment ◽

Blood Disorders ◽

Amniotic Fluid Stem Cells ◽

Fetal Survival ◽

In Utero Transplantation

Abstract Introduction Amniotic fluid stem cells (AFSC) are an appealing cell source for prenatal treatment of congenital blood disorders. They can be obtained safely during amniocentesis, are autologous to the fetus, and have shown hematopoietic potential when transplanted postnatally. Ex-vivo expansion to obtain adequate cell numbers is required for the clinical use of AFSC for in utero transplantation (IUT). The aim of this study was to assess long-term hematopoietic engraftment of freshly-isolated and cultured AFSC post IUT in mice, and compare it to that achieved following IUT of adult, bone marrow-derived hematopoietic stem cells (BM-HSC). Methods Lineage-depleted/c-Kit+ AFSC were isolated at embryonic day 13 (E13) from C57BL/6TgN(act-EGFP)OsbY01 (GFP) dams. AFSC were cultured for 7 days on mitotically-inactivated mouse embryonic fibroblasts with mouse embryonic stem cell media, and were sorted by flow cytometry (GFP+/c-Kit+) prior to IUT. BM-HSC (lineage-depleted/c-Kit+/Sca-1+) were isolated from 6-week old GFP mice. 104 AFSC (fresh or cultured) or BM-HSC (fresh) were injected intravenously into E14 C57BL/6J fetuses representing a congenic (autologous-like) IUT. Donor cell engraftment was assessed in the peripheral blood (PB) at 4 and 12 weeks of age by flow cytometry (% GFP+ cells within CD45+ population). Lineage characterization of engrafted cells was performed at both time points using antibodies against lymphoid (CD3, B220), and myeloid (CD11b, Gr-1) cell surface markers. Results are expressed as mean±SEM, and statistical analysis was performed using 1- or 2-way ANOVA with Bonferroni post-hoc tests. Experimental protocols were approved by the Institutional Animal Care and Use Committee at The Children’s Hospital of Philadelphia. Results Freshly-isolated AFSC expressed pluripotency (Oct-4, c-Myc, Klf-4) and hematopoietic markers (Sca-1+: 34.1±8.3%, CD34+: 10.6±3.1%, CD45+: 97.9±2.7%). Cultured AFSC had a doubling time of 3.2±0.2 days, and 80.7±1.6% of cells maintained c-Kit expression after 7 days of culture. c-Kit+ cultured AFSC expressed pluripotency and hematopoietic markers at similar levels as fresh AFSC. Array analysis of hematopoietic genes demonstrated significant under-expression of hematopoiesis-related transcription factors and regulators (including Gata1, Gata2, and Lmo2) in fresh and cultured AFSC compared to BM-HSC. Fetal survival rate was comparable following IUT with fresh AFSC (14/23; 60.9%), cultured AFSC (13/21; 61.9%) and BM-HSC (16/26; 61.5%) (p=0.9). Successful hematopoietic engraftment was seen at 4 weeks of age in pups that underwent fresh and cultured AFSC IUT, with comparable PB levels between groups (fresh AFSC: 21.9±1.0% vs. cultured AFSC: 20.4±1.4%; p=0.9). In contrast, IUT of BM-HSC resulted in hematopoietic micro-chimerism (engraftment: 0.04±0.02%; p<0.0001 vs. fresh and cultured AFSC). Hematopoietic engraftment was maintained in fresh and cultured AFSC groups at 12 weeks of age (fresh AFSC: 21.6±1.1% vs. cultured AFSC: 19.4±2.4%; p=0.9), and donor cell characterization demonstrated multi-lineage hematopoietic differentiation, with distribution similar to that of host cells (p=0.8). Conclusions Congenic (autologous-like) IUT of AFSC results in successful multi-lineage hematopoietic engraftment in immune-competent mice, which is superior to that achieved with BM-HSC. AFSC can be expanded in vitro without differentiation, and the hematopoietic potential of cultured cells post IUT is similar to that achieved with freshly-isolated AFSC. Combined with gene therapy, IUT of autologous AFSC could be a promising approach for prenatal treatment of congenital blood disorders. Disclosures No relevant conflicts of interest to declare.

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