Variations in Umbilical Cord Hematopoietic and Mesenchymal Stem Cells With Bronchopulmonary Dysplasia.

Objective: To test the hypothesis that umbilical cord blood-derived CD34+ hematopoietic stem cells (HPSC), cord tissue-derived CD90+ and CD105+ mesenchymal stem cells (MSC) vary with bronchopulmonary dysplasia (BPD). Methods: We conducted a prospective longitudinal study at a large birth center (Prentice Women's Hospital in Chicago, IL). Premature infants (N = 200) were enrolled in 2:1:1 ratio based on gestational age (GA): mildly preterm (31-32 weeks), moderately preterm (29-30 weeks), and extremely preterm (23-28 weeks). Cord blood (CB) and cord tissues (CT) were collected at birth using commercial banking kits, and analyzed for collection blood volume, tissue mass, CD34+, CD90+, CD105+ counts, and concentrations. Multiplex immunoassay was used to measure 12 cytokines and growth factors in CB plasma of 74 patients. BPD severity was defined according to NIH consensus definitions. Univariate and multivariate regression models were used to identify perinatal covariates and assess associations between stem cell concentrations, cytokines, and BPD outcomes. Results: Of 200 patients enrolled (mean GA = 30 ± 2 weeks), 30 developed mild, 24 moderate, and 19 severe BPD. Concentrations of HPSC and MSC, as measured by %CD34+, %CD90+, and %CD105+ of total cells, increased with degree of prematurity. Collection parameters varied with GA, birth weight (BW), gender, prolonged rupture of membranes, mode of delivery, chorioamnionitis, and multiple gestation. Moderate-severe BPD or death was increased with lower GA, BW, Apgar scores, and documented delayed cord clamping. %CD34+ and %CD90+ were increased with BPD and directly correlated with BPD severity. Severe BPD was positively associated with %CD34+ (beta-coefficient = 0.9; 95% CI = 0.4-1.5; P < 0.01) and %CD90+ (beta-coefficient = 0.4; 95% CI = 0.2-0.6; P < 0.001) after adjustment for covariates. CB plasma granulocyte-colony stimulating factor (G-CSF) was inversely associated with %CD90+, and decreased with BPD. Below median G-CSF combined with elevated %CD90+ predicted BPD (positive predictive value = 100%). Conclusions: CB and CT collections yielded high concentrations of HPSCs and MSCs in BPD infants, accompanied by low circulating G-CSF. These variations suggest possible mechanisms by which stem cell differentiation and function predict BPD.

A Rapid Method for Label-Free Enrichment of Rare Trophoblast Cells from Cervical Samples.

Extravillous trophoblasts (EVTs) have the potential to provide the entire fetal genome for prenatal testing. Previous studies have demonstrated the presence of EVTs in the cervical canal and the ability to retrieve a small quantity of these cells by cervical sampling. However, these small quantities of trophoblasts are far outnumbered by the population of cervical cells in the sample, making isolation of the trophoblasts challenging. We have developed a method to enrich trophoblast cells from a cervical sample using differential settling of the cells in polystyrene wells. We tested the addition of small quantities of JEG-3 trophoblast cell line cells into clinical samples from standard Pap tests taken at 5 to 20 weeks of gestation to determine the optimal work flow. We observed that a 4 min incubation in the capture wells led to a maximum in JEG-3 cell settling on the surface (71 ± 10% of the initial amount added) with the removal of 91 ± 3% of the cervical cell population, leading to a 700% enrichment in JEG-3 cells. We hypothesized that settling of mucus in the cervical sample affects the separation. Finally, we performed a proof-of-concept study using our work flow and CyteFinder cell picking to verify enrichment and pick individual JEG-3 and trophoblast cells free of cervical cells. Ultimately, this work provides a rapid, facile, and cost-effective method for enriching native trophoblasts from cervical samples for use in subsequent non-invasive prenatal testing using methods including single cell picking.

Microfluidic Immiscible Phase Filtration System for the Isolation of Small Numbers of Cells from Whole Blood.

Isolation of circulating tumor cells (CTCs) has generated clinical and academic interest due to the important role that CTCs play in cancer metastasis and diagnosis. Here, we present a PDMS and glass prototype of a microfluidic device for the immunomagnetic, immiscible phase filtration based capture, and isolation of MCF-7 breast cancer cells, from various sample matrices including PBS-based buffer, blood plasma, and unprocessed whole blood. Following optimization of surface energy of an oil-water interface, microfluidic geometry, and bead-binding kinematics, our microfluidic device achieved 95 ± 4% recovery of target cells from PBS-based buffer with 95% purity, 90 ± 3% recovery of target cells from blood plasma and recovery of ~70 ± 5% from unprocessed whole blood with purity >99% with 1 ml blood samples with 1,000 spiked target cells. From quantitative studies to assess the nonspecific carryover of contaminants from whole blood, we found that our system accomplishes a >175 fold depletion in platelets, >900 fold depletion in erythrocytes, and >1,700 fold depletion in leukocytes with respect to unprocessed whole blood, enabling us to avoid sample pre-processing. In addition, we found that ~95% of the isolated target cells were viable, making them suitable for subsequent molecular and cellular studies. We quantify and propose mechanisms for the carryover of platelet, erythrocyte, and leukocyte contamination in purified samples, rather than relying on sample pre-processing. These results validate the continued study of our platform for extraction of CTCs from patient samples and other rare cell isolation applications. © 2019 International Society for Advancement of Cytometry.

Expansion of Human Tregs from Cryopreserved Umbilical Cord Blood for GMP-Compliant Autologous Adoptive Cell Transfer Therapy.

Umbilical cord blood is a traditional and convenient source of cells for hematopoietic stem cell transplantation. Thymic regulatory T cells (Tregs) are also present in cord blood, and there is growing interest in the use of autologous Tregs to provide a low-risk, fully human leukocyte antigen (HLA)-matched cell product for treating autoimmune diseases, such as type 1 diabetes. Here, we describe a good manufacturing practice (GMP)-compatible Treg expansion protocol using fluorescence-activated cell sorting, resulting in a mean 2,092-fold expansion of Tregs over a 16-day culture for a median yield of 1.26 × 109 Tregs from single-donor cryopreserved units. The resulting Tregs passed prior clinical trial release criteria for Treg purity and sterility, including additional rigorous assessments of FOXP3 and Helios expression and epigenetic analysis of the FOXP3 Treg-specific demethylated region (TSDR). Compared with expanded adult peripheral blood Tregs, expanded cord blood Tregs remained more naive, as assessed by continued expression of CD45RA, produced reduced IFN-γ following activation, and effectively inhibited responder T cell proliferation. Immunosequencing of the T cell receptor revealed a remarkably diverse receptor repertoire within cord blood Tregs that was maintained following in vitro expansion. These data support the feasibility of generating GMP-compliant Tregs from cord blood for adoptive cell transfer therapies and highlight potential advantages in terms of safety, phenotypic stability, autoantigen specificity, and tissue distribution.

Effects of human umbilical cord blood mononuclear cells on respiratory system mechanics in a murine model of neonatal lung injury.

BACKGROUND: Mononuclear cells (MNCs) have well-documented beneficial effects in a wide range of adult pulmonary diseases. The effects of human umbilical cord blood-derived MNCs on neonatal lung injury, highly relevant for potential autologous application in preterm newborns at risk for bronchopulmonary dysplasia (BPD), remain incompletely established. The aim of this study was to determine the long-term morphologic and functional effects of systemically delivered MNCs in a murine model of neonatal lung injury. MATERIALS AND METHODS: MNCs from cryopreserved cord blood (1 × 106 cells per pup) were given intravenously to newborn mice exposed to 90% O2 from birth; controls received cord blood total nucleated cells (TNCs) or granular cells, or equal volume vehicle buffer (sham controls). In order to avoid immune rejection, we used SCID mice as recipients. Lung mechanics (flexiVent™), engraftment, growth, and alveolarization were evaluated eight weeks postinfusion. RESULTS: Systemic MNC administration to hyperoxia-exposed newborn mice resulted in significant attenuation of methacholine-induced airway hyperreactivity, leading to reduction of central airway resistance to normoxic levels. These bronchial effects were associated with mild improvement of alveolarization, lung compliance, and elastance. TNCs had no effects on alveolar remodeling and were associated with worsened methacholine-induced bronchial hyperreactivity. Granular cell administration resulted in a marked morphologic and functional emphysematous phenotype, associated with high mortality. Pulmonary donor cell engraftment was sporadic in all groups. CONCLUSIONS: These results suggest that cord blood MNCs may have a cell type-specific role in therapy of pulmonary conditions characterized by increased airway resistance, such as BPD and asthma. Future studies need to determine the active MNC subtype(s), their mechanisms of action, and optimal purification methods to minimize granular cell contamination.

PI3kα and STAT1 Interplay Regulates Human Mesenchymal Stem Cell Immune Polarization.

The immunomodulatory capacity of mesenchymal stem cells (MSCs) is critical for their use in therapeutic applications. MSC response to specific inflammatory cues allows them to switch between a proinflammatory (MSC1) or anti-inflammatory (MSC2) phenotype. Regulatory mechanisms controlling this switch remain to be defined. One characteristic feature of MSC2 is their ability to respond to IFNγ with induction of indoleamine 2,3-dioxygenase (IDO), representing the key immunoregulatory molecule released by human MSC. Here, we show that STAT1 and PI3Kα pathways interplay regulates IFNγ-induced IDO production in MSC. Chemical phosphoinositide 3-kinase (PI3K) pan-inhibition, PI3Kα-specific inhibition or shRNA knockdown diminished IFNγ-induced IDO production. This effect involved PI3Kα-mediated upregulation of STAT1 protein levels and phosphorylation at Ser727. Overexpression of STAT1 or of a constitutively active PI3Kα mutant failed to induce basal IDO production, but shifted MSC into an MSC2-like phenotype by strongly enhancing IDO production in response to IFNγ as compared to controls. STAT1 overexpression strongly enhanced MSC-mediated T-cell suppression. The same effect could be induced using short-term pretreatment of MSC with a chemical inhibitor of the counter player of PI3K, phosphatase and tensin homolog. Finally, downregulation of STAT1 abrogated the immunosuppressive capacity of MSC. Our results for the first time identify critical upstream signals for the induced production of IDO in MSCs that could be manipulated therapeutically to enhance their immunosuppressive phenotype.

Valproic acid enhances the engraftability of human umbilical cord blood hematopoietic stem cells expanded under serum-free conditions.

Valproic acid (VPA) is a histone deacetylase inhibitor previously shown to promote the proliferation and self-renewal of CD34(+) hematopoietic cells. We tested the effect of VPA in conjunction with the selective amplification technology developed by Viacell Inc. Stem cells enriched from frozen cord blood were cultured for 7 d, subjected to reselection and grown in fresh medium for a further 7 d. Treatment with VPA resulted in an average two-fold higher expansion of CD45(+)34(+) cells compared with control. Furthermore, VPA-treatment induced higher numbers of CD45(+)34(+) cells to reside in the S phase than control cultured cells and resulted in a 2.5-fold upregulation in HOXB4 expression. Importantly, VPA-treated cells reconstituted hematopoiesis in non-obese diabetic/severe combined immunodeficient mice with a six-fold higher efficiency than control cells. Collectively, our results indicate that VPA, already used clinically for neurologic disorder treatment, is a useful additive for the ex vivo culture of hematopoietic stem/progenitor cells to enhance engraftment efficiency.

Expanded CD34+ human umbilical cord blood cells generate multiple lymphohematopoietic lineages in NOD-scid IL2rgamma(null) mice.

Umbilical cord blood (UCB) is increasingly being used for human hematopoietic stem cell (HSC) transplantation in children but often requires pooling multiple cords to obtain sufficient numbers for transplantation in adults. To overcome this limitation, we have used an ex vivo two-week culture system to expand the number of hematopoietic CD34(+) cells in cord blood. To assess the in vivo function of these expanded CD34(+) cells, cultured human UCB containing 1 x 10(6) CD34(+) cells were transplanted into conditioned NOD-scid IL2rgamma(null) mice. The expanded CD34(+) cells displayed short- and long-term repopulating cell activity. The cultured human cells differentiated into myeloid, B-lymphoid, and erythroid lineages, but not T lymphocytes. Administration of human recombinant TNFalpha to recipient mice immediately prior to transplantation promoted human thymocyte and T-cell development. These T cells proliferated vigorously in response to TCR cross-linking by anti-CD3 antibody. Engrafted TNFalpha-treated mice generated antibodies in response to T-dependent and T-independent immunization, which was enhanced when mice were co-treated with the B cell cytokine BLyS. Ex vivo expanded CD34(+) human UCB cells have the capacity to generate multiple hematopoietic lineages and a functional human immune system upon transplantation into TNFalpha-treated NOD-scid IL2rgamma(null) mice.

Enhanced in vivo homing of uncultured and selectively amplified cord blood CD34+ cells by cotransplantation with cord blood-derived unrestricted somatic stem cells.

Mesenchymal stem cells have been implicated as playing an important role in stem cell engraftment. Recently, a new pluripotent population of umbilical cord blood (UCB) cells, unrestricted somatic stem cells (USSCs), with intrinsic and directable potential to develop into mesodermal, endodermal, and ectodermal fates, has been identified. In this study, we evaluated the capacity of ex vivo expanded USSCs to influence the homing of UCB-derived CD34(+) cells into the marrow and spleen of nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice. USSCs induced a significant enhancement of CD34(+) cell homing to both bone marrow and spleen (2.2 +/- 0.3- and 2.4 +/- 0.6-fold, respectively; p < .05), with a magnitude similar to that induced by USSCs that had been thawed prior to transplantation. The effect of USSCs was dose-dependent and detectable at USSC:CD34(+) ratios of 1:1 and above. Enhanced marrow homing by USSCs was unaltered by extensive culture passaging of the cells, as similar enhancement was observed for both early-passage (passage 5 [p5]) and late-passage (p10) USSCs. The homing effect of USSCs was also reflected in an increased proportion of NOD/SCID mice exhibiting significant human cell engraftment 6 weeks after transplantation, with a similar distribution of myeloid and lymphoid components. USSCs enhanced the homing of cellular products of ex vivo expanded UCB lineage-negative (lin(-)) cells, generated in 14-day cultures by Selective Amplification. The relative proportion of homing CD34(+) cells within the culture-expanded cell population was unaltered by USSC cotransplantation. Production of stromal-derived factor-1 (SDF-1) by USSCs was detected by both gene expression and protein released into culture media of these cells. Knockdown of SDF-1 production by USSCs using lentiviral-SiRNA led to a significant (p < .05) reduction in USSC-mediated enhancement of CD34(+) homing. Our findings thus suggest a clinical potential for using USSCs in facilitating homing and engraftment for cord blood transplant recipients.

Human umbilical cord blood cells differentiate into muscle in sjl muscular dystrophy mice.

Limb girdle muscular dystrophy type 2B form (LGMD-2B) and Miyoshi myopathy (MM) are both caused by mutations in the dysferlin (dysf) gene. In this study, we used dysferlin-deficient sjl mice as a mouse model to study cell therapy for LGMD-2B and MM. A single-blind study evaluated the therapeutic potential of human umbilical cord blood (HUCB) as a source of myogenic progenitor stem cells. Three groups of donor cells were used: unfractionated mononuclear HUCB cells, HUCB subfractionated to enrich for cells that were negative for lineage surface markers (LIN(-)) and substantially enriched for the CD34 surface marker (CD34(+)), and irradiated control spleen cells. We administrated 1 x 10(6) donor cells to each animal intravenously and euthanized them at different time points (1-12 weeks) after transplantation. All animals were immunosuppressed (FK506 and leflunomide) from the day before the injection until the time of euthanasia. Immunohistochemical analyses documented that a small number of human cells from the whole HUCB and LIN(-)CD34(+/-)-enriched HUCB subgroups engraft in the recipient muscle to express both dysferlin and human-specific dystrophin at 12 weeks after transplantation. We conclude that myogenic progenitor cells are present in the HUCB, that they can disseminate into muscle after intravenous administration, and that they are capable of myogenic differentiation in host muscle.

Functional cloning of IGFBP-3 from human microvascular endothelial cells reveals its novel role in promoting proliferation of primitive CD34+CD38- hematopoietic cells in vitro.

Ex vivo expansion of hematopoietic stem cells (HSCs) has been investigated as a means of enhancing engraftment of transplantation therapies, but current ex vivo expansion methods typically result in a loss of functional stem cell activity. Factors that can selectively expand human HSCs remain elusive. Recently we have isolated three functionally distinct human brain microvascular endothelial cells (HBMVECs) that differ greatly in their ability to support in vitro proliferation of human umbilical cord blood (UBC) CD34+CD38-cells. Using these distinct HBMVEC populations, we have devised a cell-based functional cloning assay to identify a molecule(s) capable of facilitating expansion of HSCs in vitro. A gene encoded for IGFBP-3 (insulin-like growth factor binding protein-3) has been identified. IGFBP-3 mRNA and protein are differentially expressed in distinct HBMVEC populations. In vitro cell proliferation assay and CD34+CD38- immunophenotype analysis showed that the addition of an exogenous IGFBP-3 to cultures of purified CD34+/-CD38-Lin- cells (CD2/CD3/CD14/CD16/CD19/CD24/CD56/CD66b/GlyA depleted) enhanced proliferation of primitive hematopoietic cells with CD34+CD38- phenotype, suggesting that IGFBP-3 is capable of expanding primitive human blood cells. These expanded primitive blood cells were illustrated to maintain ability to generate functional progenitors. IGFBP-3 belongs to a family of high-affinity IGFBPs, which binds to IGFs and modulates their actions. IGFBP-3 appears to have intrinsic bioactivity that is independent of IGF binding. We are currently exploring the underlying mechanism by which IGFBP-3 modulates proliferation of primitive hematopoietic cells, and the potential of IGFBP-3 to expand pluripotent human repopulating cells capable of hematopoietic reconstitution of irradiated NOD/SCID recipients.