Isolation and Ex Vivo Expansion of Human Hematopoietic Stem Cells Derived from Umbilical Cord Blood

Umbilical cord blood (UCB) is a current major source of hematopoietic stem cells (HSCs) for cell transplantation therapy. Cell transplantation with HSCs derived from UCB is advantageous over transplantation with HSCs from adult tissues. However, the low number of HSC derived from a single unit of UCB limits its application. Thus, ex vivo expansion is a good option to create more UCB HSCs for clinical application. The strategies for HSC expansion in vitro focus on mimicking the composition and structure of HSC natural niche by enhancing self-renewal and inhibiting lineage differentiation of HSCs. In the past decade, the mechanisms of the interaction between HSC and the natural niche have been deeply investigated. This great progress in basic research has led to advancements in UCB HSC ex vivo expansion. In addition, the biological characteristics of the originally isolated UCB HSCs correlate with outcome of subsequent ex vivo expansion. In this paper, we summarize the late progress achieved in isolation and ex vivo expansion of UCB HSCs. Importantly, we attempt to provide an impact and practicable procedure to expand UCB HSC in vitro from isolation of original HSCs to identification of expanded HSCs.


Introduction
Transplantation of hematopoietic stem cells is an important curative therapy for hematologic malignancies and nonmalignant hematologic disorders [1]. UCB has been established as a major source of HSCs for cell transplantation-based therapies [2] [3]. Compared to HSCs from adult donors, UCB HSCs are more advantageous as they have: less stringent HLA matching requirements, lower risk of graft-versus-host disease (GVHD) [4], lower donor-recipient viral transmission risk, lower relapse rate, and higher survival rate. However, transplantation using HSCs derived from UCB has only been applied in pediatric patients due to the limited number of HSC obtained from a single unit of UCB [5] [6].
Attempts to expand UCB HSC in order to enable its applicability in all patients are still continuing.
A comprehensive procedure for UCB HSC expansion includes: UCB collection, HSC isolation, HSC ex vivo expansion and character identification of expanded HSC. The biological characteristics of the obtained HSCs are critical in determining the outcome characteristics of the ex vivo expanded HSCs [7] [8].
UCB collection and HSC isolation procedure affect the biological activity of unexpanded HSCs. UCB collection was suggested to carry out on delivery when the placenta is still unexpelled, the uterine paroxysmal contraction pressure increases volume of the collected cord blood [9]. The intermittent and gentle shaking of the blood bag during the collection process can prevent the formation of blood clots [9] [10]. Cyclosporine A (CSA), an inhibitor of reactive oxygen species (ROS), is used to maintain renewal property and inhibit lineage differentiation of the HSCs by ameliorating oxygen shock/stress (EPHOSS) [1].
Human HSCs have the capacity of massive self-renewal division in vivo [11] [12]. The robust in vivo expansion of HSC was demonstrated by HSC transplantation. However, HSC ex vivo expansion still encounters many challenges. HSC expansion in vivo is strictly controlled by HSC niche [11] [13]. The strategies for HSC ex vivo expansion aimed to mimic HSC niche in vivo [14] [15]. But the knowledge about composition and structure of HSC niche in vivo remains limited. The cell growth factors and cell development factors used in HSC ex vivo expansion were successfully identified from HSC niche cells [8] [16] [17] [18].
However, the cocktail of those protein factors was proclaimed to not support HSC self-renewal ex vivo [19]. This conclusion indicated that HSC self-renewal not only depends on the growth and development factors but also depends on the clues out of the protein factors. In the past ten years, chemical library screening based on HSC biological characteristics has identified some useful chemical compounds in HSC ex vivo expansion [20]. Among of those chemicals, SR-1 and UM171 have great potential in maintaining biological characters of expanded HSCs through their synergistic effects with the growth factor panel (SCF, TPO, Flt3-ligand) [19] [20]. The optimized recipe with combination of the two chemicals and the panel of growth factors was shown to expand HSC more than 30 folds in effective engraftment [20]. The expanded HSCs by SR-1 and

Procedure
An informed consent was sought from patients before procedure commencement.
1) Record obstetric and family medical history, exclude etiology and genetic anomalies.
2) Cut the cord while the placenta is still in utero upon delivery of the term baby. Use 70% alcohol and iodine to sterilize the umbilical cord, puncture the umbilical vein, then collect UCB by the intermittent contraction of the uterus [9]. Allow continuous gentle shaking of the blood collection bag until no more blood flows out of the cord. Close the blood collection bag and place it in the icebox.
3) Collect at least 60 mls of the cord blood. This contains more than 10 8 mononuclear cells [10]. Do not allow blood clots to form. Transport the cord blood in icebox immediately to the laboratory. HSC isolation should start within 2 hours. Dilute the CSA to a final concentration of 50 μg/mL in PBS and label as solution A [1].

Isolation of Human CD34 Positive Cell
2) Dilute the umbilical cord blood in solution A at a ratio of 1:1.
3) Use a pipette to transfer 15 mL Ficoll to a sterile 50 mL centrifuge tube.
Carefully layer the diluted blood sample (30 mL) on Ficoll-Paque PLUS.   times. Suspend the cells in PBS.

Phenotypic Characterization of Isolated CD34+ Cells
6) Put the cells on a shaker before reading at the flow cytometer. The human cold blood stem cells express CD34 + CD38 -CD45RA -CD90 + CD49f + phenotype.    [30]. RSM is sequential, determining the significant factor variables, gradually eliminating the non-significant factors, and determining the optimal distance of the response value [25]. If the existing conditions are not optimal, a series of adjustments can give the optimal direction.
3) Carefully transfer HSC to a 37˚C, 5% supplemented CO 2 incubator. On day 7, perform cell count and flow cytometry analysis of CD34 positive cells.

Optimizing Concentration of the Chemical Compounds
SR-1 and UM171 are powerful chemical molecules screened from chemical library containing more than 5000 compounds [19] [20]. SR-1 is an antagonist of the aryl hydrocarbon receptor (AhR). It inhibit the AhR signaling pathway and promote the proliferation of HSC by binding AhR to prevent its binding to AhR photoaffinity ligand (PAL) [19] [21]. Conversely, UM171 enhance the human HSC self-renewal machinery independently of AhR suppression [20], whereas induce a rheostatic regulation of inflammatory and anti-inflammation/detoxification programs [33]. UM171 greatly reduces the risk of long-term frequent immune complications (GVHD) [22]. But SR-1 or UM171 could not stimulate HSC pro-

HSC Functional Analysis in Vivo-Transplantation Study
The NSG mice at aging of 8 -12 weeks are prepared for HSC transplantation by feeding with water containing antibiotics.

Conclusion
HSCs derived from umbilical cord blood have advantages inaccessible and he-

Conflicts of Interest
The authors declare no conflicts of interest regarding the publication of this paper.

Source of Support
This research was supported by Logistic research project of china (aws17j007).