Umbilical Cord Blood Hematopoietic Stem Cells Research Articles

Expansion of Umbilical Cord Blood Hematopoietic Stem Cells for Clinical Use.

Umbilical cord blood (UCB) is an alternative donor source for allogeneic hematopoietic stem cell transplantation. However, transplantation in adults is frequently limited by the small number of cells available in a unit. We have previously developed the technology to expand hematopoietic stem cells (HSC) and stromal/mesenchymal stem cells (SMSC) from all UCB frozen samples (13, 3). The incubation of thawed UCB mononuclear cells (MNC) in the presence of SCF (25ng/ml), FLt-3 (25ng/ml), MGDF (10ng/ml) & IL-6 (20ng/ml) and 10% human serum in stroma-free liquid culture not only generated long term expansion of transplantable UCB HSC (non-adherent). Also, long-term expansion of SMSC (adherent cells) was successful. In order to upscale the expansion to use for clinical applications, we analyzed 3 frozen UCB comparing the expansion from 24-well plates (previous) versus expansion in bags (VueLife™) after 10 and 14 days of culture. Results show substantial expansion in total cell count (TCC, 4.8, 10.9: 2.4, 3.8 fold) at d10 and d14 from wells & bags respectively. TCC increased further in the presence of SMSC (38% & 33% in CD34+ cell count cultured in wells). CD133+CD34+CD38- HSC multiplied (11, 25 fold, d10/d14 & colony forming cells (CFC) 19 fold at d14 bags respectively). Heterogeneous cell populations were detected after d 14 in bags: T and B -lymphoid (%CD3/%CD19; 65/4:50w/3), megakaryocytic (%CD61; 7:4) and myeloid (%CD33; 31:43) at d0/d14 respectively. Further, expanded cells (250,000) containing a small number of CD133+CD34+CD38- (15,000–30,000) were injected into the liver of sub-lethally irradiated newborn Balb/C Rag2-Cγ−/− mice. Our preliminary data show no engraftment from cells expanded in wells and bags after 6 weeks of transplantation from d10 cultures (human CD45 + <1%). However, positive engraftment in mouse PB was detected from cells expanded for 14 d (wells, 1.16–2.5% & bags, 1.21–3.9%) as compared to control mice (CD45; 30% PB & 70% BM) receiving selected CD34 + (300,000 CD34+ at d0). Primitive repopulating cells (PRC), and multilineage human CFC were detected after transplantation. On d14 of HSC expansion, UCB MNC derived adherent cells (SMSC) were enriched by trypsinization. SMSC were established in serum free and serum plus culture as well. The immunophenotype of harvested SMSC was CD29+, CD44+ and CD45−, CD34− and CD133− at percentages + >90%. Following repeated trypsinization, SMSC count increased 41–96 folds. CFU-Fibroblast colonies (92–173) were generated from 104 SMSC after 2 weeks in MesenCult medium. We have previously differentiated SMSC into hepatocytes. Now we also generated adipocytes in an induction medium containing, Insulin, dexamethasone & indomethacin. SMSC formed Oil-droplet vacuoles in the cytoplasm in 3 weeks. The culture conditions we defined to maintain UCB PRC should be developed clinically. SMSC described herein exhibit in vitro properties of multipotent stem cells.

Leukemia Inhibitory Factor (LIF): Marrow-Derived Human Mesenchymal Stem Cell (huMSC) Secretion and Potential Impact on Umbilical Cord Blood (UCB) CD133+ Hematopoietic Stem Cells (HSC) Differentiation.

In vitro expansion of UCB using cytokines has been pursued to overcome the limited stem cell content of a single UCB graft. We have previously demonstrated that a feeder layer of huMSC inhibits UCB HSC differentiation during short-term cytokine-driven expansion in vitro. The protein LIF has been shown to inhibit the differentiation of neurosphere stem cells during 3 week culture over a monolayer of murine stromal cells (C. Shih, et al., 2001). We sought to investigate the hypothesis that LIF secreted by bone marrow (BM) derived huMSCs is involved in inhibition of UCB HSC differentiation during short-term cytokine-driven expansion in vitro. BM derived huMSCs (third passage) were cultured at 2x10^6/ml in DMEM supplemented with 10% FBS. Supernatant was collected at 24, 36, 48, and 72 hours and analyzed for LIF secretion levels by ELISA (Quantikine). LIF secreted by huMSC was noted at all four time points, with peak secretion at 48 h (mean 52.1±3.3 pg/ml) (n=3). UCB was obtained according to institutional guidelines after normal full-term deliveries, collected into bags with citrate dextrose (Allegiance), and MNC were separated on a Histopaque-1077 (Sigma) density gradient. UCB CD133+ cells were isolated using AutoMACS magnetic cell sorter (Miltenyi) and surface stained for LIF receptor (LIF-R). Surface expression of LIF-R on gated CD133+ cells was 2.61%. Total LIF-R expression in isolated CD133+ cells was further confirmed by Western blot (n=3) using anti-LIF-R antibody (Chemicon). Isolated UCB CD133+ were plated in 24 well plates at 3.3x10^3/ml and cultured in StemPro™ media supplemented with 10% FBS, L-glutamine, penicillin, streptomycin and amphotericin B (Gibco). UCB CD133+ were culture-expanded for 96h with or without the addition of recombinant human LIF (Chemicon) (10ng/ml) in a combination of cytokines including: IL-3 (20 ng/mL), IL-6 (20 ng/mL), Flt-3L (100 ng/mL) (R&D), SCF (100 ng/mL), G-CSF (20 ng/mL), and EPO (0.1 U/mL) (Amgen Inc.). At 0, 48, and 96 h cell counts were obtained and flow analysis was performed including surface staining for: CD133, CD34, CD38, HLA-DR, CD33, CD71, and CD41B (Becton Dickinson). At 48 hours, higher cell counts in cultures without LIF were noted 6.3x10^4 ± 0.9/ml, compared with cultures with LIF 4.4x10^4 ± 0.8/ml. However, at 96 h cell counts equalized when comparing cultures with or without LIF at 7.510x10^4 ± 0.9/ml, and 7.8x10^4±0.9/ml respectively (n=6). Surface expression of differentiation markers on gated CD133+ cells did not differ when comparing cultures (n=3). In summary, we observed LIF secretion by MSC peaks at 48 h at a concentration 3 logs lower than that previously used to inhibit stem cell differentiation (10 ng/ml). Although LIF-R is expressed on CD133+ HSC, no difference in cell expansion nor surface phenotype of UCB 133+ cells was observed at early time points (96h) during expansion in cytokines with or without the addition of LIF. Taken together, huMSC mediated inhibition of cytokine-driven UCB HSC differentiation is not attributable to LIF secretion alone and may require direct cell contact between UCB CD133 HSC and huMSC. Studies are ongoing to determine whether LIF may augment huMSC-based UCB CD133 expansion.

Maximal Cord Blood Recovery and CD34+ Progenitor Cell Collection Using Machine Pulsatile Perfusion of Placentas.

Umbilical cord blood (UCB) is an attractive source of hematopoietic stem cells (HSC) because of greater availability, less stringent HLA matching requirements, and lower incidence and severity of GVHD. Currently, UCB transplant procedures in adults are limited by low collection volumes of total nucleated cells and CD34+ HSC. Approaches to ex vivo expand long-term engraftable HSC have been widely unsuccessful. Recent studies have clearly demonstrated that infusion of a greater number of cells UCB cells enhances the rate of engraftment and lowers the risk of transplantation-related mortality. Machine pulsatile perfusion (MPP) has been successfully used to select cadaveric renal allografts for transplantation, to isolate human islets from pancreata and shown to be a useful cardiac preservation technique in canine heart transplant studies. In this study, the feasibility of using machine pulsatile perfusion to collect human UCB total nucleated cells and CD34+ HSCs was evaluated using placentas designated for research purposes. Immediately following delivery UCB (65 ± 15 mL, n=5) was first collected by needle aspiration from the umbilical cord vein in accordance with standard procedures then followed by MPP (~500 ml) of the placental arteries within 2–3 hours of delivery. Clinically total nucleated cells count (TNC), CD34+ cell numbers and myeloid, erythroid and multipotent CFU progenitor cell content of UCB units are used as predictive measurements of hematopoietic/engraftment potential. Low-density cells (<1.077 g/mL) were isolated by density centrifugation. The median number of viable low density cells obtained was 488 × 106 (range, 240–652 × 106), and 1541 × 106 (range, 888–1800 × 106) for UCB and MPP collections, respectively. MMP low density cell preparations contained significantly more mature segmented neutrophils with a low percentage (<0.1%) of sheared-off vessel wall endothelial cells. Both UCB and MPP low density cells collections showed similar number of assayable CFU-GEMM, CFU-GM, CFU-M, and CFU-G progenitor cells. In contrast, MMP collected cells contained 2–3 times more erythroid BFU-E colonies than UCB collections. Equivalent numbers of CD34+ HSC were enumerated by FACS analysis and subsequently isolated by positive immunomagnetic MACS selection from MPP and UCB collections. Likewise, the progenitor cell content (CFU-GEMM, CFU-GM, CFU-M, CFU-G and BFU-e) of the isolated CD34+ cell populations derived from each cell collection were very similar. These results demonstrate that pulsatile perfusion can be performed easily after traditional UCB collection procedures. This technique effectively recovers on average twice as many TNC and multilineage CD34+ HSC cells when compared to traditional UCB collection procedures. Altogether these results are particular promising since increased numbers of UCB HSC available for infusion should result in accelerated hematopoietic recovery. Moreover, the demonstrated enhanced HSC cell yield together with the simplicity of collection could potentially widen the clinical applicability of UCB transplants in adults.

N-rasm-Transduced Fibroblasts Facilitate the Expansion and Engraftment of Umbilical Cord Blood Stem Cells.

Clinical trials using ex-vivo expanded umbilical cord blood (UCB) cells have thus far failed to demonstrate significant improvement in speed of hematopoietic recovery suggesting better understanding of self-renewal, proliferation, homing and engraftment capabilities of cord blood is needed. Recent progress has been made in the identification and understanding of the cellular niche for hematopoietic stem cells (HSCs) within bone marrow however attempts to identify soluble factors that regulate HSC engraftment to bone marrow have been less successful. We have demonstrated that N-rasmtransduced NIH 3T3 fibroblasts release soluble factors that support the ex-vivo expansion of UCB CD34+ hematopoietic progenitor cells (HPCs). UCB CD34+ cells were cultured in combination with cytokines : TPO, SCF, IL-6 and flt3-ligand(all at 100ng/ml). Significant (7-fold) expansion of total nucleated cells was seen on day 19 in cultures complemented with medium produced by N-rasm-transduced fibroblasts (N-rasmmedium), compared to control cells (GFP- transduced fibroblasts (GFP medium)). Maximum expansion of UCB CD34+ cells (2-fold) and the most primitive sub-population of CD34+ CD117+ cells (2-fold) were seen on day 4 in N-rasmmedium cells compared to GFP medium cells. Furthermore, increased numbers of cycling cells were observed in UCB CD34+ cell cultures supplemented with N-rasmmedium. Significant (3-fold) expansion of CD34+CXCR4+ cells enriched with the NOD/SCID mice engrafting cells, was also observed. This increased expansion of CD34+CXCR4+ cells correlated with a 3-fold increased engraftment of human UCB cells in a NOD/SCID mouse model confirming retention of engraftment capability of expanded cells. This data demonstrates that N-rasm-transduced fibroblasts secrete soluble factors that promote ex-vivo expansion and in-vivo engraftment of primitive human hematopoietic progenitor/stem cells derived from UCB. Proteins that are secreted by N-rasm-transduced fibroblasts that activate hematopoiesis are currently being identified. Our data demonstrates the potential of N-rasm-transduced fibroblasts as a reservoir for the discovery of novel secreted proteins that regulate ex-vivo expansion and engraftment of human UCB HSCs and may represent a potential novel expansion strategy allowing greater use UCB as a stem cell source for allogeneic transplantation.

Differentiation of Non-Adherent Hematopoietic Stem Cells from Umbilical Cord Blood Cells into Adherent Hepatocytic Lineage.

The liver consists of a variety of different cell types, the vast majority of which are hepatocytes. It has been shown that adult, as well as umbilical cord blood (UCB) mesenchymal stem cells (MSC) are capable of differentiation into mesenchymal and non-mesenchymal tissues including hepatocytes. Only a limited number of UCB units (20%) such as frozen, unfractionated and concentrate leukocytes induced by hydroxyethyl starch volume reduced UCB units can generate MSC. In light of this, we have developed the technology to generate potent MSC from all frozen UCB samples tested (3/3). In the present study, the incubation of thawed out UCB mononuclear cells (MNC) and non-selected CD34+ cells in the presence of SCF (25ng/ml), FLt-3 (25ng/ml), MGDF (10ng/ml) & IL-6 (20ng/ml) and 10% human serum in stroma-free liquid culture not only generated long term expansion of transplantable UCB hematopoietic stem cells (non-adherent) (Peters et al., 2004), but also long-term expansion of MSC (adherent cells). Adherent cells were enriched by medium changes followed by trypsinization and subsequent culture and passing on fibronectin in the presence of 10% human serum and 30ng/ml bFGF. The stroma-free liquid cultures were tested for the production of hematopoietic stem cells (HSC), as well as MSC over a period of 15 weeks; MSC increased throughout expansion of HSC. We used the CFU-Fibroblast assay to demonstrate MSC activity in stromal cell formation in vitro. Expanded MSC between passages 3–5 were plated using MesenCult™ (Stem Cell Technologies). After 14 days incubation at 37oC, 5% CO2, very large and small CFU-fibroblast colonies generated from 105 MNC/ml (initiated on Day 0 in stroma-free liquid culture) were continuously detected throughout expansion. In order to test the capacity of MSC to differentiate into hepatocytes, cells were cultured in DMEM supplemented with dexamethasone and 25ng/ml HGF, the latter of which was replaced with 50ng/ml oncostatin M after incubation day 14. The cells were able to expand to cell lines and took on the common hexagonal shape characteristic of hepatocytes; this parameter was used as a primary screening criterion. In addition, the hepatocyte-like cells were further assessed by reverse transcription-polymerase chain reaction and found to express mRNA for albumin, as well as two hepatocyte-specific bile salt transport proteins, basolateral bile transport system NTCP and the canalicular bile salt export pump BSEP. These data demonstrate the presence of pluripotent stem cells in UCB with the capacity to differentiate into a hepatocytic lineage.The research in this area has great potential, both with respect to future application in patients with end-stage liver disease, as well as for learning more about the role of MSC in new clinical cellular therapies.

Telomere length changes after umbilical cord blood transplant

The establishment of donor-derived hematopoiesis in the recipients of hematopoietic stem cell (HSC) transplants involves extensive proliferation and differentiation of HSCs. Data from long-term survivors of HSC transplants suggest that these transplanted HSCs may experience a debilitating replicative senescence. A significant posttransplant shortening of peripheral blood mononuclear cell (PBMNC) telomeres has been observed in both marrow transplant and peripheral blood progenitor cell transplant recipients. Similar studies have not been performed for umbilical cord blood (UCB) HSC transplants, which might be expected to exhibit increased posttransplant replicative potential due to their inherently greater telomere length. Blood was obtained from donor-recipient pairs of allogeneic PBHSC transplant and UCB HSC transplant, both before transplant and at follow-up treatments (minimum 1 year after transplant) after engraftment. Telomere restriction fragment length (TRFL) analysis was performed on the blood samples. The mean TRFL and posttransplant changes in the mean TRFL were analyzed. Measurements of telomere lengths in the PBMNCs of transplant patients revealed a significant net decrease in telomere length in all transplant recipients compared with their respective donors. Our results also revealed that the PBMNCs of umbilical cord stem cell transplant patients retain a significantly longer posttransplant telomere length. The significantly longer telomeres observed in the allogeneic UCB HSC transplant recipients compared to the allogeneic PBHSC transplant recipients in our study may be indicative of a replicative advantage inherent in the use of UCB HSC for transplant.

Enhanced In Vivo Homing of Uncultured and Selectively Amplified Umbilical Cord Blood CD34+ Cells by Co-Transplantation with Cord Blood Derived Unrestricted Somatic Stem Cells.

Mesenchymal stem cells have been implicated as playing an important role in stem cell engraftment. A new pluripotent population of umbilical cord blood (UCB)-derived Unrestricted Somatic Stem Cells (USSCs), with intrinsic and directable potential to develop into mesodermal, endodermal and ectodermal fates, has recently been identified and characterized (1). In this study we evaluated the capacity of USSCs to influence the homing of UCB -derived CD34+ cells into the marrow and spleen of NOD/SCID mice. Cultured USSCs were co-transplanted with CFDA-labeled cord blood CD34+ cells into sublethally irradiated NOD/SCID mice. Femurs and spleens were harvested 16 hrs thereafter and the percentage of CD34+ cells determined by flow cytometry.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<0.05). Similar findings were obtained with frozen USSC samples that had been thawed prior to transplantation. The effect of USSCs was specific, as no homing enhancement could be observed by co-transplantation of CD34+ cells with lineage-positive UCB cells, which contain T, B and mature myelo-erythroid cells. Enhanced marrow homing by USSCs was unaltered by extensive culture passaging of the cells, with a similar degree of enhancement observed for both early (p5) and late (p10) passage USSCs (1.7 ± 0.1 and 1.9 ± 0.1 -fold, respectively). Enhanced homing was dose-dependent, detectable at USSC/CD34+ ratios of 1:1 and above.USSCs were also found to enhance the homing of day 14 cells harvested from cultures of selectively amplifiedTM, ex-vivo expanded UCB lineage-negative (lin-) cells. The relative proportion of homing CD34+ cells within the culture-expanded cell population, was unaffected by USSC co-transplantation. Our findings thus demonstrate that USSCs enhance the homing of UCB hematopoietic stem cells and suggest a clinical potential for these cells in facilitating engraftment under conditions of limiting cord blood stem cell numbers.

Risk of Viral Transmission Via Bone Marrow Progenitor Cells Versus Umbilical Cord Blood Hematopoietic Stem Cells in Bone Marrow Transplantation

Hematopoietic stem cell transplantation (HSCT) is the treatment of choice for children and certain adults with malignant and nonmalignant hematologic disease. Since viral infections are the major problem, this study examined those that might potentially be transmitted to HSCT recipients via bone marrow (BM) versus umbilical cord blood (UCB). BM progenitor cells, peripheral blood leukocytes, and plasma samples were collected from 30 allogenic BM donors. Umbilical cord blood hematopoietic stem cells and plasma samples were also collected from 34 UCB donors. Viral DNA extracted and purified from collected specimens was processed using nested polymerase chain reactions (PCR) to detect human parvovirus B19 (HPV B19), human herpesvirus-6 (HHV-6), varicella-zoster virus (VZV), human cytomegalovirus (HCMV), and Epstein-Barr virus (EBV). The prevalences of HCMV DNA in collected BM progenitor cells versus UCB hematopoietic stem cells were 73% versus 23%, respectively. Conversely, HHV-6 DNA was not detected in any collected specimen by simple PCR. Distribution of the other investigated virus DNAs except EBV DNA was similar in specimens collected from both groups. EBV DNA was not determined in UCB hematopoietic stem cells. The results indicate that the risk of viral transmission to BM transplant recipients via UCB hematopoietic stem cells is less than that with BM progenitor cells.

Successful Expansion of Umbilical Cord Blood Cells as a Source of Hematopoietic Progenitors for Adult Transplantation.

The low yield of progenitor CD34+ cells recovered from umbilical cord blood (UCB) limits the utility of this source for transplantation in adults. This limitation has triggered investigations into how ex vivo expansion of hematopoietic stem cells (HSC) could be achieved to allow for transplantation in larger recipients. In the present study, the incubation of MNC and not selected CD34+cells in the presence of SCF 25ng/ml+MGDF 10ng/ml+FLt-3 25ng/ml+IL-6 20ng/ml, and 10% human serum in stroma-free liquid culture generated long-term expansion of transplantable UCB HSC. In vitro, HSC expansion from 13 UCB lasted >7 months giving 39, 1.3x104, and 4.7 x109 fold increase in total cell count after 14, 70 and 217 d of expansion as compared to d0 (105/ml). Similarly, CD34+ and CD34+/CD38− cell populations increased reaching 229 and 2.2x105 and 91 and 2.2x104 fold after 14 and 70d of expansion. Examination of cell morphology and analysis by flow cytometry showed the presence of primitive and mature cells belonging to all hematopoietic cell lineages. Similarly, multilineage colonies with recloning capacity were generated in culture. Erythroid, myeloid and mixed colonies increased by 116 and 1.8x104 fold and megakaryocytic colonies by 8 and 527 fold after 14 and 70d. Expanded cells were karyotypically normal and lacked the most common chromosome translocations seen in AML and CML t (15,17) and t (9, 22). HSC expanded for 6 and 13 weeks and cropreserved for 6–11 months were able to re-expand in liquid culture and generate colonies capable of recloning and multi-lineage differentiation. We estimated the frequency of SCID repopulating cells (SRC) in UCB samples expanded for 2 and 12 w using 1000,500,250 and 125 unselected CD34+ cells injected intravenously into sublethally irradiated NOD/SCID mice. Mice were sacrificed 20 weeks after transplantation. Human cell engraftment measured as CD45+ (HuCD45+) was detected in all mice (x3mice/dilution) (0.1–9.8%). In addition, HuCD45+ cells with multilineage phenotype were present, (CD19 (lymphoid), CD33 (myeloid), CD71and Glycophorin-A (erythroid) as well as CD34+/CD38− cells). SRC increased by 90 fold after 2 weeks of expansion and by 187 fold after 12 weeks compared to unexpanded CD34+cells. Additional proof of human cell engraftment was documented using semisolid culture (MethoCultTM GF H4434 Stem Cell Technologies). Human myeloid and erythroid colonies were generated from all dilutions, and counts ranged between 63–271/500,000 MNC.Initial studies to test the relative magnitude of UCB HSC expansion from 24-well plates to culture bags (OptiCyte TM, Baxter) using one UCB, the total cell count increased by (6.3 and 20 (bags) Vs 2.3 and 3.3 fold (wells) after 7 and 14d) and CD34+ subpopulations including CD34+/CD38−. Based on these ongoing results, a phase II clinical trial using ex vivo expanded UCB for 14d in a setting of sub ablative Conditioning is planned.

CD34 expression on long-term repopulating hematopoietic stem cells changes during developmental stages

AbstractThe CD34 antigen serves as an important marker for primitive hematopoietic cells in therapeutic transplantation of hematopoietic stem cells (HSC) and gene therapy, but it has remained an open question as to whether or not most HSC express CD34. Using a competitive long-term reconstitution assay, the results of this study confirm developmental changes in CD34 expression on murine HSC. In fetuses and neonates, CD34 was expressed on Lin−c-Kit+ long-term repopulating HSC of bone marrow (BM), liver, and spleen. However, CD34 expression on HSC decreased with aging, and in mice older than 10 weeks, HSC were most enriched in the Lin−c-Kit+CD34− marrow cell fraction. A second transplantation was performed from primary recipients who were transplanted with neonatal Lin−c-Kit+ CD34high HSC marrow. Although donor-type HSC resided in CD34-expressing cell fraction in BM cells of the first recipients 4 weeks after the first transplantation, the stem cell activity had shifted to Lin−c-Kit+CD34− cells after 16 weeks, indicating that adult Lin−c-Kit+CD34− HSC are the progeny of neonatal CD34-expresssing HSC. Assays for colony-forming cells showed that hematopoietic progenitor cells, unlike HSC, continue to express CD34 throughout murine development. The present findings are important because the clinical application of HSC can be extended, in particular as related to CD34-enriched HSC and umbilical cord blood HSC.

Retrovirally Transduced CD34++ Human Cord Blood Cells Generate T Cells Expressing High Levels of the Retroviral Encoded Green Fluorescent Protein Marker In Vitro

Human umbilical cord blood (UCB) hematopoietic stem cells (HSC) receive increased attention as a possible target for gene-transfer in gene therapy trials. Diseases affecting the lymphoid lineage, as adenosine deaminase (ADA) deficiency and acquired immunodeficiency syndrome (AIDS) could be cured by gene therapy. However, the T-cell progenitor potential of these HSC after gene-transfer is largely unknown and was up to now not testable in vitro. We show here that highly purified CD34++ Lineage marker-negative (CD34++Lin-) UCB cells generate T, natural killer (NK), and dendritic cells in a severe combined immunodeficient mouse fetal thymus organ culture (FTOC). CD34++Lin- and CD34++CD38-Lin- UCB cells express the retroviral encoded marker gene Green Fluorescent Protein (GFP) after in vitro transduction with MFG-GFP retroviral supernatant. Transduced cells were still capable of generating T, NK, and dendritic cells in the FTOC, all expressing high levels of GFP under control of the Moloney murine leukemia virus (MoMuLV) long terminal repeat promotor. We thus present an in vitro assay for thymic T-cell development out of transduced UCB HSC, using GFP as a marker gene.

Hematopoietic Potential of Cryopreserved and Ex Vivo Manipulated Umbilical Cord Blood Progenitor Cells Evaluated In Vitro and In Vivo

The hematopoietic potential of cryopreserved and ex vivo manipulated umbilical cord blood (UCB) samples was evaluated in vitro and in vivo. Phenotypic analysis shows that approximately 1% of cord blood mononuclear cells express high levels of CD34 antigen on their surface (CD34hi), but none of a panel of lineage antigens (Lin-), suggesting that they are hematopoietic progenitor cells that have not yet committed to a specific lineage. Approximately 1% of CD34hi/Lin- cells are primitive hematopoietic progenitors that produce B lymphoid and multiple myeloid progeny for up to 7 weeks in stromal cell cultures. Twenty-one percent (+/- 13%) of CD34hi/Lin- cells also express low levels of the Thy-1 antigen and are threefold to fourfold enriched over CD34hi/Lin- cells in primitive hematopoietic potential as measured by long-term culture and phenotypic analysis. One-week liquid cultures of CD34-enriched UCB progenitor cells in the presence of interleukin (IL)-3, IL-6, and stem cell factor (SCF) results in a two-fold to threefold expansion of progenitors capable of reinitiating long-term stromal cell cultures. Only the CD34hi/Thy-1+/Lin- cell population was capable of maintaining progenitors with secondary transfer potential in long-term stromal cell cultures and is thus postulated to contain all of the primitive hematopoietic stem cells in UCB. The in vivo transplantation potential of UCB was also measured. Ex vivo manipulated UCB progenitor cells were used to engraft irradiated human thymus fragments implanted in severe combined immunodeficiency (SCID) mice. Thymic engraftment with >5% donor-derived cells and a normal CD4/CD8 distribution was observed in 19 of 23 tissues tested. UCB cells from in vitro expansion cultures engrafted with efficiencies comparable to nonexpanded cells. Similar results were obtained for UCB engraftment of human bone fragments implanted in SCID mice. In all cases, engraftment was achieved in competition with endogenous competitor stem cells and across major histocompatibility barriers. Taken together, this data demonstrates that human UCB is a rich source of multipotent hematopoietic progenitors that can be cryopreserved, enriched by physical methods, and expanded in a limited fashion without measurable loss of long-term culture or in vivo engrafting potential as measured in these assays.