Abstract

INTRODUCTION

Cord blood is a valuable source of hematopoietic stem cells and alternative for bone marrow transplantation. The first transplantation of cord blood was carried out in 1988 ¹. At 2013 more than 30000 transplantations were accomplished ².

Umbilical cord blood contains similar cell populations to bone marrow and peripheral blood mobilized stem cell products: hematopoietic stem cells, progenitor cells and differentiated functional cells. The total cell number in the cord blood transplant is lower compared to other sources. Bone marrow and peripheral blood mobilized stem cell grafts should contain approximately 10⁸ CD34+ cells, while cord blood graft only 5 x 10⁶ CD34+ cells. The number of nucleated cells per kg of weight of the patients correlates with outcome. The minimal dose of transplanted cord blood total nucleated cells (TNCs) for hematological disorders is 20-25 million/kg weight ³. It was suggested that a minimum cryopreserved TNC dose in cord blood transplant should be 2.5 x 10⁷ cells/kg for a single unit with 5/6 matched graft, and 5 x 10⁷ cells/kg for a single-unit 4/6 matched graft in patients with malignancies ⁴. But when no grafts with higher cell doses or other stem cell sources are available, cord blood grafts containing 1,0- 2.0 x 10⁷cells/kg could be useful ⁵. In comparison, for bone marrow transplantation 1.5-3 x 10⁸ nucleated cells/kg and for peripheral blood mobilized cells 2-7 x 10⁸ mononuclear cells/kg are recommended ⁶. Therefore, cord blood hematopoietic stem cells (HSCs) seem to be more potent than HSCs from adult bone marrow and peripheral mobilized blood. There does not appear to be any long-term engraftment problems to recipients of cord blood, though they have delayed neutrophil and platelet engraftment in comparison to bone marrow transplanted patients ⁷ , ⁸.

The cord blood stem cells also survive in culture longer and proliferate better. It was demonstrated that cord blood CD34+ cell expansion in cultures was 31-250-times better than the expansion of bone marrow CD34+ cells over the same culture conditions and period. A higher incidence of colony-forming cells with greater proliferation capacity in cord blood over bone marrow CD34+ cells was also reported ⁹.

Nonobese-diabetic severe combined immunodeficient (NOD/SCID) mice transplanted with cord blood mononuclear cells (MNCs) showed significantly higher levels of chimerism than those transplanted with bone marrow MNCs ¹⁰. When transplanted in vivo, cord blood has greater repopulating ability than bone marrow or mobilized peripheral blood. The greater repopulating ability of cord blood is due to the higher generative ability of cord blood HSCs ¹¹. According some researchers, the main cause of delayed neutrophil and platelet recovery after cord blood transplantation (for neutrophils – mean 18 days in bone marrow transplantation group and mean 32 days in cord blood transplantation group, for platelets: mean 29 days and mean 81 days, respectively), is the low number of short-term engrafting cells or mature progenitor cells ¹² , ¹³.There are clinical evidences that patients who receive higher doses of TNC and CD34⁺ cells,  engraft faster than do patients who receive lower doses ¹⁴.The ex vivo expansion of cord blood cells is important way to overcome the relatively low number of cells in cord blood unit.

This paper reviews the preclinical experiments of cord blood HSCs expansion and clinical studies with use of ex vivo expanded cord blood.

Rationale for Hematopoietic Stem Cell Expansion

Maintenance of stem cell pool throughout the life requires the asymmetric division, when one progeny cell retains stemness and other differentiates. Ex vivo expansion of stem cells requires a symmetric division, when both daughter cells should maintain properties of stem cells. Correct balance of cytokines, medium, sera and other active substances used for expansion is extremely important. The final goal of cord blood expansion is to generate clinically safe, transplantable HSC’s, which can repopulate recipient. The acceptable alternative could be ex vivo generation of more mature progenitors, which would be able to engraft rapidly.

The initial efforts on HSC expansion were done in the 1970s. Researchers used bone marrow in liquid culture system ¹⁵ , ¹⁶ , ¹⁷ , ¹⁸. Bone marrow-derived adherent cells supported prolonged proliferation and differentiation of genetically incompatible stem and precursor cells. In the similar systems with prosimians normal hematopoiesis has been sustained in vitro for more than 1 year ¹⁹. Many efforts were done to expand cells in order to retain stemness versus differentiation with use of either cytokines or stromal cells, or their combination. Usage of stromal cell mimicries stem cell niche and specific microenvironment, necessary for cell survival and proliferation. The long-term engraftment and subsequent reconstitution of normal hematopoiesis occur if the HSCs home to specialized niches of the bone marrow. HSCs do not grow as autonomous units. They are surrounded by the marrow microenvironment, defined by the cell-cell interactions, cell-extracellular matrix interactions and by the growth factors. The bone marrow microenvironment contains fibroblasts, smooth muscle cells, endothelial cells and mesenchymal stem cells. They provide a scaffold to the stem cells and produce transmembrane ligands, extracellular matrix components and soluble proteins. HSCs are located in the endosteal osteoblastic niche attached to spindle-shaped N-cadherin+ CD45- osteoblastic (SNO) cells. The homing is a crucial moment of transplantation. Two processes are important: stem cells adhesion to the endothelial cells and migration across them into niche after adhering to the vessel wall. Adhesive molecules fix HSCs to their niche. They also provide signals to quiescent HSCs in an undifferentiated state – in G0 stage. Growth factors start HSC cell cycle entry. The SNO cells provide inhibitory signals (BMP, Jagged and Ang-1) to protect HSCs from differentiation and apoptosis. HSCs express very late activation antigen-4 (VLA-4 or CD49d), VLA-5 (CD49e) and lymphocyte function associated antigen-1 (LFA-1 or CD11a), which adhere to vascular cell adhesion molecule-1 (VCAM) and fibronectin of SNO. The non-integrin adhesion receptor, lymphocyte homing associated adhesion molecule (HCAM or CD44) and stromal cell-derived factor (SDF-1) also play important roles in engraftment ²⁰ , ²¹ , ²².

Growth Factors

The cytokines in cell culture system can have influence on total nucleated cell and stem cell expansion, long-term donor engraftment and leukocytes and platelets recovery speed. The most important cytokines and their optimal concentration for HSC blood stem cell expansion were determined ²³. Most protocols of cord blood expansion involve stem cell factor (SCF), thrombopoietin (TPO), IL-3, IL-6, Flt3-ligand. In stroma-free culture systems SCF and Flt3-ligand maintain HSCs ²⁴. TPO supports the expansion of both short- and long-term repopulating HSCs. They can be significantly expanded, rather than maintained ²⁵. Cytokine components affect recovery of different cell lineages ²⁶.

The Action of Individual Cytokines and Their Combinations on Cord Blood Stem Cell Expansion

Stem cell factor (SCF) or c-kit ligand. This cytokine is a hematopoietic growth factor and exerts its activity by signalling through the c-Kit receptor. Up to 3% of cord blood cells express C-kit (tyrosine-protein kinase kit, CD117) ²⁷. Treating of cord blood CD34+ stem cells with SCF improved homing of cord blood cells in NOD/SCID mice models. The delayed hematopoietic reconstitution after umbilical cord blood transplantation could be result of significantly lower levels of CD49e, CD49f, and CXCR-4 on cord blood CD34+ cells than on counterparts from peripheral blood and bone marrow ²⁸. Short-term treatment of cord blood-derived CD34+ cells with SCF resulted in upregulation of the homing-related molecules and increased ex vivo transmigratory and in vivo homing potential of cord blood stem cells ²⁹.

Flt3-ligand. This cytokine regulates early hematopoietic cell proliferation. It binds to the hematopoietic cells expressing the tyrosine kinase receptor Flt3/flk2. It was demonstrated that Flt3-ligand is essential for expansion of the early stem cells (CD34+ Thy-1+ CD45+) ³⁰ and enhances adhesiveness by activation and increased expression of CD49d and CD49e, which are important in proliferation and differentiation ³¹.

Thrombopoietin (TPO) has an important role in regulation of primitive hematopoietic cells. TPO signals through the c-mpl receptor. HSCs from fetal liver, cord blood, bone marrow and mobilized peripheral blood express c-mpl on their surface at high density ³². The addition of TPO as a single cytokine to bone marrow hematopoietic cells had dramatic effects and resulted in the generation of both long- and short-term repopulating HSCs ³³.

IL-6. This cytokine plays an important role in host defense by regulating immune and inflammatory responses. IL-6 also acts in synergy with SCF and Flt3-ligand. IL-6 maintains immature phenotype of hematopoietic stem cells. The addition of IL-6 to SCF, Flt3-L, TPO, IL-3 combination caused better ex vivo expansion of cord blood CD133+ cells ³⁴.

IL-3 is a growth factor that promotes the survival, differentiation and proliferation of different lineages of committed progenitors. IL-3 increases total expanded cell count, expands human mature blood cells and hematopoietic progenitor cells (HPCs), but has an inhibitory effect on the expansion of long-term-repopulating HSCs and often were reported that this cytokine can play a negative role in primitive cell expansion. It was demonstrated that TPO + SCF + Flt3-ligand expanded mobilized peripheral blood CD34+ fraction maintain multilineage engraftment ability in NOD/SCID mice, which is abrogated by the addition of IL-3 ³⁵ , ³⁶ , ³⁷.

Severe Combined Immunodeficiency (SCID) Mice Repopulating Cell (SRC) Increase after Cord Blood Expansion

The number of SRC is the crucial parameter for the predicting of the repopulating ability of native or expanded human hematopoietic stem/progenitor cells after transplantation into patients receiving high-dose radio- or chemotherapy. The SRCs represent very primitive hematopoietic cells and are exclusively CD34+CD38-, while colony forming cells (CFC) and long-term culture initiating cells (LTC-IC) could be found also in the CD34+CD38+ fraction ³⁸ , ³⁹ , ⁴⁰.

To find the cytokine combination for the stem cell expansion without change in repopulating potential, CD34+CD38-Lin- cord blood cells were cultured with several cytokines. It was demonstrated that SCF+ Flt3-ligand is the minimal necessary growth factor combination and TPO can replace IL-3, IL-6 and G-CSF without changing. Though TNC expansion and CD34+ cell percentage were different for each cytokine cocktail: SCF+Flt3-ligand+IL-3+IL-6+G-CSF (447-fold and 22.5%, respectively), for SCF + Flt3-ligand combination (14-fold and 48.8%, respectively) and for SCF + Flt3-ligand + TPO combination (241-fold and 29.7%, respectively), the transplantation of cultured cells in each condition resulted in multilineage hematopoietic repopulation in NOD/SCID mice and all protocols supported similar frequencies and levels of human hematopoietic engraftment ⁴¹.

Expanded human cord blood CD34+ cells (with cytokine combination: SCF+Flt3- ligand+TPO+complex IL-6₊soluble IL-6 receptor) were successfully repopulated in NOD/SCID mice. After 10–12 weeks of transplantation in the bone marrow of 13 from 16 transplanted mice the human CD45+ cells were detected. The proportion of CD45+ cells in bone marrow of expanded cell recipient animals was 11.5% of all bone marrow cells – 10 times higher than in those receiving comparable numbers of fresh CD34+ cells⁴².

Sublethally irradiated NOD/SCID  mice were transplanted with cryopreserved unmanipulated CD34+ cord blood cells or expanded CD34+ cord blood cells. For up to 10 weeks expanded cells were engrafted better in comparison with the same numbers of unmanipulated cells. The combination of SCF (50 ng/ml) + Flt3-ligand (50 ng/ml) + TPO (20 ng/ml) + IL-6 (10 ng/ml) was superior. In this combination SRCs are expanded 960-fold after 9 to 10 weeks of expansion⁴³. In another study, on the feeder cells (AFT024) plus Flt3-ligand + SCF + IL-7 or Flt3-ligand + TPO expanded CD34+ cord blood cells were engrafted in primary, secondary and tertiary xenogeneic hosts: NOD/SCID mice or fetal sheep. SRCs with multilineage differentiation potential were maintained in both systems for up to 28 days. Marrow from mice was highly engrafted by expanded cells, induced multilineage human hematopoiesis in 50% even in secondary recipients. Cells, which were expanded for one week, engrafted primary, secondary, and tertiary fetal sheep ⁴⁴.

Apoptosis, Telomere Length and Telomerases Activities in Expanded Cord Blood Cells

The apoptosis rate during expansion depends on cytokine combination. In expansion study on mobilized peripheral blood CD34+ Thy-1+ cells the combination of SCF, TPO and Flt3-ligand resulted in the less number of apoptotic cells and slight increase of CD34+ Thy-1+ cells, while leukemia inhibitory factor (LIF) along with IL-3 and IL-6 reduced the number of CD34+ Thy-1+ cells to 30% of initial count ⁴⁵. Telomerase activity and telomere length were studied in the long-term culture expanded CD34+ cells. Cord blood cells were cultured either on adenovector transduced stromal cells, expressing TPO, or stimulated by a cytokines (SCF, FL, TPO and IL-6) without stromal cells. Telomere length in the cultivated CD34⁺ cell population in the first 6 weeks of expansion was increased, remained stable up to 16 week and then diminished slightly. The telomerase activity in the first weeks was also increased,thereafter decreased ⁴⁶.

THE SURFACE PHENOTYPE OF EXPANDED STEM CELLS

The surface phenotype of stem cells during expansion can be changed because of action of growth factors and may not give fully adequate information on the cells. The expression of cell surface antigens and receptors, responsible for cell migration and adhesion, could be altered during expansion and, theoretically, could cause change of the homing properties and engraftment of expanded cells. But several experimental data showed that homing potential of expanded cells remained unaltered. It was reported that one-week ex-vivo expansion at least preserves cord blood stem cell homing-related characteristics and migration activities.Isolated CD34+ cells from the fresh or cryopreserved haematopoietic stem cells were expanded with Flt3-ligand, SCF and TPO ± IL-3 cocktail. Most freshly purified CD34+ cells expressed CD49d and CD44. The expression of these receptors on the expanded CD34+ cells was increased or remained similar to the fresh cord blood CD34+ cells. Both spontaneous and SDF-1-induced adhesions were highest on day 10 and then declined on day 14. Spontaneous migration quickly decreased in the second week of expansion. On day 14 expanded CD34+ cells showed hardly any spontaneous migration over filters. The SDF-1-induced migration on day 10 was comparable to that on day 0, but was significantly lower on day 14 ⁴⁷.

Karyotype and Gene Expression of Expanded Cells

There are data that genetic characteristics, at least karyotype, of expanded cord blood stem cells (CD34+/CD38-) after 20 and 43 days in culture, are preserved ⁴⁸.

Gene-expression of expanded cord blood stem cells was determined by the Affymetrix microarray system in study on influence of collagen I presence on cord blood CD34+ cell expansion. The significant differences in the molecular signature between suspension cultures and HSC cultures with collagen gel were found. Upregulation of many genes, including a high number of cytokines or chemokines: IL-8, IL-1α, IL-1b, TNF-α and MIP-1α, which have influence on the proliferation and mobilization of HSCs, was detected. Collagen imitated the HSC niche microenvironment and induced the secretion of mediators by HSCs and/or progenitor cells ⁴⁹.

Preclinical Studies oN Cord Blood Expansion and Clinical Trials of Expanded Cord Blood Transplantation

Results of some preclinical studies on cord blood expansion are shown in Table 1.

The first attempt to expand cord blood haematopoietic stem cells was done by Hal Broxmeyer in 1992. Umbilical cord blood mononuclear cells were expanded in Iscove’s modified Dulbeccos’s medium (IMDM) with FBS and SCF alone or with either IL-3, GM-CSF, G-CSF or Epo for 7 days. In the experiment with SCF alone CFU-GM was increased 7.9 ± 1.1-fold, with SCF plus any other cytokines – 12.5 ± 1.4-fold, BFU-E – respectively 2.2 ± 0.2-fold and 3.6 ± 0.7-fold, CFU-GEMM – respectively 2.7 ± 0.4-fold and 3.5 ± 0.4-fold ⁵⁰. Later several groups developed stroma-free culture systems supplemented with multiple early acting cytokines. In long-term culture system purified cord blood CD34+ cells were cultured in IMDM supplemented with FCS and several cytokines (IL-3, IL-6, SCF, Flt3-ligand, alone or in a combination). The best result was achieved by using of combination Flt3-ligand (50 ng/ml) plus TPO (10 U/ml). This combination was capable of sustaining for more than 6 months proliferation and renewal of cord blood primitive stem cells. In the long-term culture-initiating cells assay (LTC-IC) expanded primitive stem cell number at week 25 was increased 83886-fold, total cell number –50,000,000-fold, CD34+ and CD34+CD38-cells were expanded in the similar ways ⁵¹.

Cord blood stem cells were expanded with the different ligands for Notch pathway activation. Notch signaling is a crucial for the onset of definitive hematopoiesis in the embryos ⁵². Notch-1 receptor ligands had influence on CD34+ cell expression in human cord blood. Lower densities of Notch ligand Delta1 enhanced the generation of human cord blood stem CD34+ cells, while the higher densities resulted apoptosis of CD34+ cells. Notch-mediated ex vivo expansion system was developed, which markedly enhanced the absolute number of cord blood CD34+ cells and their repopulation ability in the marrow of NOD-SCID mice ⁵³. Recently Delaney and colleagues developed clinically relevant Notch-mediated ex vivo expansion system for human CD34+ cord blood progenitors in StemSpan serum-free medium with IL-3, IL-6, TPO, Flt3-ligand and SCF. Cultivation resulted in a marked increase of absolute number of stem/progenitor cells, which were infused in ten patients with high-risk acute leukemia after a myeloablative preparative regimen. The time to neutrophil recovery was shortened – in a phase I trial transplantation the median time for neutrophil recovery was 16 days instead of expected 23 to 26 days ⁵⁴.

De Lima et al ⁵⁵ transplanted patients with AML/MDS (n=21), ALL (n=6), NHL (n=2), CLL (n=2), and HD (n=1) with expanded cord blood cells. Double transplantation of unmanipulated and expanded cord blood units, matched in at least 4/6 HLA antigens, with a minimum of 1×10⁷ TNC/kg per unit was carried out. For 14 days cord blood was expanded on confluent layer of mesenchymal stem cells (MSCs) together with cytokines: SCF, Flt-3 ligand, G-CSF, and TPO. The expanded cells were infused along with a second unmanipulated cord blood unit. The median number of TNC and CD34+ cells/kg in unmanipulated cord blood was 2.35 × 10⁷ and 0.95 × 10⁵, respectively. The median number of expanded cells and CD34+ cells/kg was 5.8 × 10⁷  and 8.7 × 10⁵, respectively. A median expansion of cord blood cells was 14-fold for TNC and 40-fold for the CD34+ cells. A median time to neutrophil and platelet engraftment of 15 (range 9-42) and 40 (range 13-62) days, respectively were reported. After 21 days chimerism analysis demonstrated that cells from expanded units were significantly represented in recipients: 19%, 16% and 14% of mononuclear, T lymphocyte and myeloid cell populations were derived from expanded donor cells. One-year survival was 40%. This method led to fast engraftment of neutrophils and platelets, and high-rates of platelet transfusion independence.

Cord blood was expanded with the copper chelator tetraethylenepentamine (TEPA), which diminishes cord blood stem cell differentiation and augments the expansion potential of cord blood-derived progenitor cells, because cellular copper supports stem cell differentiation. TEPA along with early acting cytokines (TPO, IL-6, Flt3-ligand and SCF) resulted in a proliferation of mature, committed cord blood cells (CD34+Lin+) and colony-forming units cells (CFUc) as well as robust expansion of the CD34+CD38- and CD34+Lin- subpopulations ⁵⁶ , ⁵⁷. Human progenitor cells, expanded with TEPA, engrafted significantly better in SCID mice, than the same number of the unmanipulated or without TEPA expanded cells ⁵⁸ , ⁵⁹. After preclinical studies I/II phase clinical trial was launched. Cord blood CD133+ cells were expanded with TEPA and cytokines for 21 days and transplanted in acute leukaemia and lymphoma patients. The unexpanded cells of the same cord blood units were infused 24 h earlier. Cord blood units and patients were matched at 4 or 5 HLA loci. Though the median total number of infused TNC per kg weight was only 1.8 x 10⁷, 90% of patients engrafted, no severe GVHD was reported. Netrophil and platelet engraftment time was 30 and 48 days, respectively, and 100-day survival was 90% ⁶⁰.

Could the expanded cord blood overcome the delayed engraftment of cells, particularly in adult patients? Abilities of expanded cord blood in shortening time to engraftment and reducing the graft failure rate were studied in one clinical trial, where ex vivo expanded cord blood were transplanted in 37 patients. 12 children and 25 adults (23 of them had progressive refractory disease at study entry), with hematologic malignancies (34 patients) or breast cancer (3 patients) treated with high-dose chemotherapy followed by cord blood transplantation, matched or mismatched at 1 or 2 alleles. No minimal cord blood cell dose requirement was established, though attempts were made to obtain cord blood which had ≥1 x 10⁷ TNC/kg in unit. A cord blood CD34+ cells were expanded for 10 days in medium with SCF, G-CSF and TPO. Median expansion was 4-fold. The remainder of the cord blood graft was infused without further manipulation. Each patient was transplanted with a median of 0.99 x 10⁷ TNC/kg (together expanded and unexpanded). The median engraftment time for neutrophil was 28 days, for platelets – 106 days. Extensive acute and chronic GVHDs were described in 40% and 63% of patients, respectively. After 30 months 35% of patients survived, among children 58% event-free survival rate was documented, which was similar to matched sibling donor transplantation survival rate ⁶¹.

In the other study, patients with AML/MDS (N=25; 35%), ALL (N=17; 24%), NHL (N=10; 14%), HD (N=7; 10%), CML (N=5; 7%), and CLL (N=7; 10%) were transplanted with double cord blood unit. The majority of cord blood units were 4/6 HLA matched. From the smallest unit selected CD133+ cells were cultured for 14 days in media containing SCF, G-CSF and TPO.  The T cell-containing CD133-negative fraction was frozen. Median TNC expansion was 23-fold, CD34+ cell  expansion – 2.3-fold. On day 0, the unmanipulated unit was infused, followed by the CD133-negative  and the expanded fractions. Infused median TNC was 3.6 x 10⁷/kg and median CD34+ cell count – 1.8 x10⁵/kg. Patients, who received expanded cells and a reduced-intensity regimen, engrafted neutrophils in a median of 7 days (range, 4-15 days; n=14) versus 14 days (range, 5-32 days; n=12) in the unmanipulated transplantation in another 36 patient (p = 0.05). Thirty-four patients were alive (48%) with a median follow up of 11.3 months. Twenty-four patients relapsed (34%). Chimerism analysis showed that ultimately one cord blood unit dominated in all patients, on both the unmanipulated and expanded arms of the trial. The majority of patients had evidence of expanded cord blood posttransplant chimerism (7-82%). In half of those patients, the expanded cord blood unit predominated over the unmanipulated unit for 2-12 months posttransplant (52-87%), followed by gradual predominance of the unmanipulated cord blood unit by 14 months in all patients. One and two years survival in patients of expanded arm were better than unmanipulated arm: 1-year survival rate was 60% and 46% (p = 0.1) and 2-years survival – 55% and 20%, respectively ⁶². 28 patients were transplanted with expanded allogeneic cord blood at the Duke University, North Carolina. The largest part of cord blood were transplanted unmanipulated (1×10⁷/kg), the rest (1-2×10⁸ cells) were expanded with AastromReplicell Cell system for 12 days in IMDM with PIXY321 (granulocyte-macrophage colony stimulating factor/interleukin-3 fusion protein), EPO and Flt3-ligand. The median fold expansion was of 2.4. The median fold expansion for CFU-GMs was 82.7, for CD34+ cells – 0.5. 27 patients received ex vivo-expanded cells. Patients with malignant (19 patients) or non-malignant disorders were transplanted with 1, 2, or 3 alleles mismatched cells. Augmentation of cord blood transplants with ex vivo-expanded cells did not alter the time to myeloid, erythroid or platelet engraftment in 21 evaluable patients. Recipients of ex vivo-expanded cells had durable engraftment with a median follow-up of 47 months (range: 41-51 months). The infusion of expanded cells was safe ⁶³.

Expansion with Components of the Hematopoietic Microenvironment

Ex vivo cord blood expansion removes stem cells from the microenvironment and needs the addition of cytokines for proliferation. Alternatively, cord blood cells could be cocultured with components of the hematopoietic microenvironment. Ex vivo contact with stromal components of the hematopoietic microenvironment preserves different activity of HSC. It was shown that bone marrow endothelial cells (ECs) are essential for reconstitution of hematopoiesis due to Notch signaling pathway and support long-term expansion of HSC ⁶⁴. Endothelial and perivascular cells maintain HSCs in bone marrow niche also because of SCF secretion ⁶⁵.

Expansion of cord blood CD34+ cells with SCF, Flt3-ligand, IL-6, VEGF and IL-1, with or without autologous coculture system using umbilical vein endothelial cells (HUVEC) demonstrated that coculture system was superior over liquid culture: after 10 days of cultivation the total cell number increased 77-fold and 10-fold, respectively; CD34+ cell number, coculture with HUVEC, was increased 23-fold, while in liquid culture – only 3-fold ⁶⁶. Recently was reported cord blood CD34+ cell expansion on HUVEC, modified by introducing a lentiviral vector expressing E4ORF1 gene of adenovirus – E4+EC. This gene enabled long-term survival of endothelial cells in serum-free/growth factor-free conditions. Coculture of cord blood CD34+ cells in direct cellular contact with hECs and minimal concentrations of TPO/Kit-ligand/Flt3-ligand (50 ng/ml each) resulted in a 400-fold expansion of TNCs and 150-fold expansion of CD45+/CD34+progenitor cells over a 12-day period, while cytokines alone resulted in 125-fold expansion of TNCs and 50-fold expansion of CD45+/CD34+ cells. Long-term engraftment of cocultivated cord blood in NSG mice was better than with cytokine expanded cord blood cells ⁶⁷. The same group has compared E4+EC from HUVEC and mesenhymal stem cell from placenta as a feeder layers for cord blood CD34+ expansion with SCF, TPO and FLT-3 Ligand. In this study both types of feeder layers supported hematopoietic expansion. Endothelial cells triggered amplification of CD34+/CD38−/CD90+ cells, they differentiated into all cell lineages and were capable of engraftment and multi-lineage differentiation in sub-lethally irradiated mice. Mesenchymal progenitors promoted amplification of CD34+CD38+ cells, defined as precursors with more limited differentiation potential ⁶⁸.

MSCs have clinical immunomodulatory activity and influence on graft versus-host disease in transplant patients, build the components of the stem cell niche. In combination with cytokines, MSCs can increase the cord blood hematopoietic progenitor expansion, because provide components for the stem cell niche, which are absent in common liquid cultures. MSCs abolish the need for positive selection, can improve expansion and engraftment. The hematopoietic microenvironment is composed of hematopoietic and non-hematopoietic components, which direct self-renewal and proliferation of stem cells and regulate the differentiation and maturation. Ex vivo contact with MSCs preserve HSC activity. MSCs can be isolated and expanded as adherent cells from a variety of fetal and adult tissues, including cryopreserved cord blood. The efficient expansion protocol has been established for cord blood MSCs (DMEM with human AB serum, Flt-3-ligand, SCF, TPO and IL-6), which enabled long-term expansion. MSC stemness and their differentiation capacity in vitro were preserved throughout long-term culture. After 24 passages 95% of expanded MSCs displayed a fibroblast-like morphology, expressed MSC-related antigens (CD105, CD73, CD29, CD44, CD133), were negative for haematopoietic cell markers (CD45, CD34 and CD140 ⁶⁹. Another source for MSCs is Wharton’s jelly stromal cells after enzymatic ⁷⁰ or nonenzymatic isolation ⁷¹. Wharton’s jelly MSC transplantation along with cord blood HSCs from the same donor could play important role for cell-based therapy in the future and their combined banking should be considered ⁷² , ⁷³.

In several cord blood expansion clinical protocols CD133+ or CD34+ cells are isolated before culture. After this procedure remarkable number of HSCs, as well as huge number of TNC, are lost and even after successful expansion, the final number of cells, available for transplantation, is quite low ⁷⁴. To show the advantage of unmanipulated cord blood expansion over selected stem cell expansion, two methods were compared: CD133+-selected cord blood cells were expanded in αMEM medium with 10% FBS, and G-CSF, SCF, TPO. After 14 days of culture, cells were harvested and assayed for TNC, CFU, CD133+ and CD34+. Bone marrow MSCs were isolated from healthy donor marrow and cultured in a αMEM supplemented with FBS. After 2-3 days adherent cell culture achieved approximately 70% confluence. Non-selected cord blood, containing initially  10⁹ TNC, were expanded in medium with 20% FBS and G-CSF, SCF and TPO and plated over the pre-established confluent MSC monolayer for 14 days. Up to 10¹⁰ TNC were generated. In liquid culture, initiated with the CD133+-selected cord blood cells, after 14 days of expansion, TNC number were increased from 20 mln to 800 mln. Cord blood MSC coculture also generated a significantly greater output of CD133+ cells (>7-fold, p = 0.01), CD34+ cells (>14-fold, p = 0.003), than did ex vivo liquid culture of CD133+-selected cord blood. MSC coculture with cord blood cells without a selection step and thus, without loosing of stem cells, might produce cord blood graft with a better engraftment potential. Cotransplantation of unmanipulated cord blood with expanded cord blood, cocultured with MSCs, could result in a combined graft, containing both hematopoietic progenitor cells, responsible for long-term repopulation (mostly derived from the unmanipulated unit), and in cells, responsible for short-term repopulation (derived from with MSCs cocultured cord blood unit). Such a combination of cord blood units potentially provides the recipient with more rapid and long-term engraftment ⁷⁵.

Wharton’s jelly-derived MSCs supported ex vivo expansion of cord blood CD34+ cells in the absence of exogenous cytokines. After 7 and 14 days coculture the expansion rate of the CD34+ cells was increased by 2.0 and 7.3 times, respectively. In particular, CD34+/CD90+ and CD34+/CD33- populations were increased. Expanded cells were transplanted in NOD/SCID mice. After 8 weeks human CD45+ cell percentage in mouse bone marrow was evaluated as engraftment marker. For 7 days expanded cells engrafted better than unmanipulated cells ⁷⁶.

New Cytokines

New cytokines were used for the preclinical studies. Three new growth factors, secreted by the endothelium – Angiopoietin-like 5 (Angptl5), IGF-binding protein 2 (IGFBP2) and pleiotrophin in combination with conventional cytokines enhanced HSC expansion. Pleiotrophin is an 18-kDa heparin-binding growth factor. Its addition to the combination of TPO, SCF and Flt3-ligand significantly increased CD34+/38-/Lin- cell number. Immunodeficient mice in three different experiments were transplanted with uncultured human cord blood CD34+/38-/Lin- cells and with expanded CD34+/38-/Lin- progenies, cultured with or without pleiotrophin. At 4 and 8 weeks after transplantation with pleiotrophin expanded cells significantly improved engraftment of NOD/SCID mice in comparison to transplantation with uncultured CD34+CD38−Lin−cells or with the progeny of cells cultured with TPO, SCF and Flt3-ligand alone. Thus, pleiotrophin increased number of SRCs among cord blood stem cells ⁷⁷.

Addition of angiopoietin-like 5 or IGF-binding protein 2 to SCF, TPO, FGF-1 in a serum-free culture led to a sizable expansion of HSC numbers, assayed by NOD/SCID transplantation: 20-fold expansion of SRCs in cord blood CD133+ cell culture was reported ⁷⁸.

3D Strategy

Most of the current hematopoietic culture systems use two dimensional expansion strategies for cell expansion, even if these systems mimicry niches by use of MSCs or endothelial cells. But in reality the niche is three dimensional, where stem cells are interacting with extracellular matrix and several types of supportive cell. The usage of 3D polymers for HSC expansion has been studied recently. 3D scaffold increases the cell growth area, provides structural support, and improves cell-to-cell contact, thus fulfills the important requirements of the niche. Cord blood CD34+ cells were expanded on different type of 3D biomaterial scaffolds (polycaprolactone (PCL), poly-lactic-co-glycolic acid (PLGA), fibrin and collagen) in combination with umbilical cord MSCs and cytokine cocktail in StemSpan medium: SCF, TPO, FGF-1, angiopoietin like-5(Angptl-5), IGFBP2. Expanded cells were transplanted into NSG mice. All scaffolds but PLGA supported cord blood CD34+ cell expansion. Cells in fibrin scaffold reached the highest growth (5 × 10⁸-fold expansion of total nuclear cells after fourteen days and 3 × 10⁷-fold expansion of CD34+ cells after seven days), maintained a more primitive immunophenotype for more cell divisions and exhibited superior migratory and adhesive properties, showed the significantly higher numbers of engraftment and multilineage differentiation (CD45, CD34, CD13, CD3 and CD19) in bone marrow, spleen and peripheral blood in long-term transplanted NSG mice compared to the other 3D biomaterial scaffolds ⁷⁹. Currently cord blood HSCs were expanded on extracellular matrix, synthesized after decellularization of ex vivo expanded stromal cell structure. In such system with standard cytokines (TPO, Flt3-ligand, SCF and IL-6 in Stemline II medium) HSC expansion rate reached 85-fold after 7 days ⁸⁰.

Conclusions

Studies for cord blood HSC expansion for clinical use will be actively continued in the near future. The development of the new growth factors, cytokines, biologically active substances and additives for expansion of stem cells, which maintain their undifferentiated condition, the new biopolymers for stem cell expansion on 3D scaffolds, which mimicry HSC niche, new media for optimal expansion of cord blood HSCs, coculture with autologous or allogeneic supportive cells (MSCs from different sources: cord blood, amnion, chorion, Wharton’s jelly; endothelial cells), culture in the new bioreactors with improved pH, O₂, CO₂, glucose control, feeding, aeration and agitation control, will improve stem cell expansion outcome.

The generation of routine expansion method for optimal number of cord blood HSCs, which are clinically safe, engraft long-term and rapidly recover recipients bone morrow in several days is the main goal of cord blood expansion. The developments of the last decade made this goal more realistic than ever before.

Conflict of Interest

The Authors declare that they have no conflict of interests. No financial support was received from any source for the manuscript.

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