Functional Stability (at +4°C) of Hematopoietic Stem and Progenitor Cells Amplified Ex Vivo from Cord Blood CD34+Cells

Mesenchymal stem cells (MSCs) are known to support the characteristic properties of hematopoietic stem and progenitor cells (HSPCs) in the bone marrow hematopoietic microenvironment. MSCs are used in coculture systems as a feeder layer for the ex vivo expansion of umbilical cord blood (CB) to increase the relatively low number of HSPCs in CB. Findings increasingly suggest that MSC-derived microvesicles (MSC-MVs) play an important role in the biological functions of their parent cells. We speculate that MSC-MVs may recapitulate the hematopoiesis-supporting effects of their parent cells. In the current study, we found MSC-MVs containing microRNAs that are involved in the regulation of hematopoiesis. We also demonstrated that MSC-MVs could improve the expansion of CB-derived mononuclear cells and CD34+cells and generate a greater number of primitive progenitor cells in vitro. Additionally, when MSC-MVs were added to the CB-MSC coculture system, they could improve the hematopoiesis-supporting effects of MSCs. These findings highlight the role of MSC-MVs in the ex vivo expansion of CB, which may offer a promising therapeutic approach in CB transplantation.

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Ex Vivo Expansion of Cord Blood Hematopoietic Stem and Progenitor Cells in a Wharton’s Jelly and Bone Marrow Stromal Cell Based Co-Culture System

Blood ◽

10.1182/blood.v124.21.1129.1129 ◽

2014 ◽

Vol 124 (21) ◽

pp. 1129-1129

Author(s):

Dandan Li ◽

Omar S. Aljitawi ◽

Richard A. Hopkins

Keyword(s):

Bone Marrow ◽

Cord Blood ◽

Progenitor Cells ◽

Stromal Cell ◽

Bone Marrow Stromal Cell ◽

Hematopoietic Stem ◽

Marrow Stromal Cell ◽

Cd34 Cells ◽

Stem And Progenitor Cells ◽

Cord Blood Cd34

Abstract Umbilical cord blood (UCB) is of great value by providing transplantable hematopoietic stem and progenitor cells (HSPCs). Compared with HSPCs from adult bone marrow and periferial blood, UCB cells are more primitive with higher proliferation ability, and UCB HSPC transplantation requires less HLA matching. The major problems in UCB transplantation is the limited number of transplantable cells in each unit which are often insufficient for transplantation in adults In order to elucidate the effects of the main components of bone marrow on cord blood CD34+ expansion, freshly enriched cord blood CD34+ cells were cultured in contact with bone marrow stromal cell (BM-MSC) monolayer, BM-MSCs pre-seeded in decellularized Wharton's jelly matrix (DWJM), and in DWJM alone, as well as separated by a transwell preventing the physical contact between CD34+ cells and BM-MSCs in a medium supplemented with a cytokine cocktail including Flt-3 igand, stem cell factor, and thrombopoietin. Expansion patterns were analyzed by CD34+ and CD45+ expansion, as well as colony-forming unit (CFU) assay. We found that the number of CFU increases significantly in cord blood CD34+ cells co-cultured with BM-MSCs in DWJM compared to that of control. Particularly, the increase in CFU-GEMM, BFU-E and CFU-GM number is more significant compared to other colonies. Importantly, DWJM alone is not able to increase CFU numbers compared to that of the control indicating there is a synergistic effect between DWJM and BM-MSCs on cell stemness. Surprisingly, BM-MSCs in DWJM also increses CD34+ cell expansion by 2 to 3 fold after one week culture, presumably from enhanced ablilty of self-renewal from CD34+ cells. Therefore, our data suggest DWJM synergizes with BM-MSCs to increase CD34 cell stemness, which can potentially be used in the clinic therapy. Disclosures No relevant conflicts of interest to declare.

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Clinical-scale validation of a new efficient procedure for cryopreservation of ex vivo expanded cord blood hematopoietic stem and progenitor cells

Cytotherapy ◽

10.1016/j.jcyt.2016.08.004 ◽

2016 ◽

Vol 18 (12) ◽

pp. 1543-1547 ◽

Cited By ~ 1

Author(s):

Pascale Duchez ◽

Laura Rodriguez ◽

Jean Chevaleyre ◽

Philippe Brunet De La Grange ◽

Zoran Ivanovic

Keyword(s):

Cord Blood ◽

Progenitor Cells ◽

Scale Validation ◽

Ex Vivo ◽

Clinical Scale ◽

Hematopoietic Stem ◽

Efficient Procedure ◽

Stem And Progenitor Cells

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Directed Differentiation of Mobilized Hematopoietic Stem and Progenitor Cells into Functional NK Cells with Enhanced Antitumor Activity

Cells ◽

10.3390/cells9040811 ◽

2020 ◽

Vol 9 (4) ◽

pp. 811

Author(s):

Pranav Oberoi ◽

Kathrina Kamenjarin ◽

Jose Francisco Villena Ossa ◽

Barbara Uherek ◽

Halvard Bönig ◽

...

Keyword(s):

Nk Cells ◽

Progenitor Cells ◽

Peripheral Blood ◽

Cell Expansion ◽

Nk Cell ◽

Ex Vivo ◽

Feeder Cells ◽

Hematopoietic Stem ◽

Cd34 Cells ◽

Stem And Progenitor Cells

Obtaining sufficient numbers of functional natural killer (NK) cells is crucial for the success of NK-cell-based adoptive immunotherapies. While expansion from peripheral blood (PB) is the current method of choice, ex vivo generation of NK cells from hematopoietic stem and progenitor cells (HSCs) may constitute an attractive alternative. Thereby, HSCs mobilized into peripheral blood (PB-CD34+) represent a valuable starting material, but the rather poor and donor-dependent differentiation of isolated PB-CD34+ cells into NK cells observed in earlier studies still represents a major hurdle. Here, we report a refined approach based on ex vivo culture of PB-CD34+ cells with optimized cytokine cocktails that reliably generates functionally mature NK cells, as assessed by analyzing NK-cell-associated surface markers and cytotoxicity. To further enhance NK cell expansion, we generated K562 feeder cells co-expressing 4-1BB ligand and membrane-anchored IL-15 and IL-21. Co-culture of PB-derived NK cells and NK cells that were ex-vivo-differentiated from HSCs with these feeder cells dramatically improved NK cell expansion, and fully compensated for donor-to-donor variability observed during only cytokine-based propagation. Our findings suggest mobilized PB-CD34+ cells expanded and differentiated according to this two-step protocol as a promising source for the generation of allogeneic NK cells for adoptive cancer immunotherapy.

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Ex-Vivo Expansion of Human Cord Blood CD34+ Cells by Overexpression of Bmi-1.

Blood ◽

10.1182/blood.v108.11.1329.1329 ◽

2006 ◽

Vol 108 (11) ◽

pp. 1329-1329

Author(s):

Aleksandra Rizo ◽

Edo Vellenga ◽

Gerald de Haan ◽

Jan Jacob Schuringa

Keyword(s):

Cord Blood ◽

Cell Expansion ◽

Cultured Cells ◽

Ex Vivo ◽

Human Cord Blood ◽

Self Renewal ◽

Hematopoietic Stem ◽

Cd34 Cells ◽

Cord Blood Cd34

Abstract Hematopoietic stem cells (HSCs) are able to self-renew and differentiate into cells of all hematopoietic lineages. Because of this unique property, they are used for HSC transplantations and could serve as a potential source of cells for future gene therapy. However, the difficulty to expand or even maintain HSCs ex vivo has been a major limitation for their clinical applications. Here, we report that overexpression of the Polycomb group gene Bmi-1 in human cord blood-derived HSCs can potentially overcome this limitation as stem/progenitor cells could be maintained in liquid culture conditions for over 16 weeks. In mouse studies, it has been reported that increased expression of Bmi-1 promotes HSC self-renewal, while loss-of-function analysis revealed that Bmi-1 is implicated in maintenance of the hematopoietic stem cells (HSC). In a clinically more relevant model, using human cord blood CD34+ cells, we have established a long-term ex-vivo expansion method by stable overexpression of the Bmi-1 gene. Bmi-1-transduced cells proliferated in liquid cultures supplemented with 20% serum, SCF, TPO, Flt3 ligand, IL3 and IL6 for more than 4 months, with a cumulative cell expansion of more then 2×105-fold. The cells remained cytokine-dependent, while about 4% continued to express CD34 for over 20 weeks of culture. The cultured cells retained their progenitor activity throughout the long-term expansion protocol. The colony-forming units (CFUs) were present at a frequency of ~ 30 colonies per 10 000 cells 16 weeks after culture and consisted of CFU-GM, BFU-E and high numbers of CFU-GEMM type progenitors. After plating the transduced cells in co-cultures with the stromal cell line MS5, Bmi-1 cells showed a proliferative advantage as compared to control cells, with a cumulative cell expansion of 44,9 fold. The non-adherent cells from the co-cultures gave rise to higher numbers of colonies of all types (~70 colonies/10.000 cells) after 4 weeks of co-culture. The LTC-IC frequencies were 5-fold higher in the Bmi-1-transduced cells compared to control cells (1/361 v.s. 1/2077, respectively). Further studies will be focused on in-vivo transplantation of the long-term cultured cells in NOD/SCID mice to test their repopulating capacity. In conclusion, our data implicate Bmi-1 as an important modulator of human HSC self-renewal and suggest that it can be a potential target for therapeutic manipulation of human HSCs.

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Activation of OCT4 enhances ex vivo expansion of human cord blood hematopoietic stem and progenitor cells by regulating HOXB4 expression

Leukemia ◽

10.1038/leu.2015.189 ◽

2015 ◽

Vol 30 (1) ◽

pp. 144-153 ◽

Cited By ~ 29

Author(s):

X Huang ◽

M-R Lee ◽

S Cooper ◽

G Hangoc ◽

K-S Hong ◽

...

Keyword(s):

Cord Blood ◽

Progenitor Cells ◽

Ex Vivo ◽

Ex Vivo Expansion ◽

Human Cord Blood ◽

Hematopoietic Stem ◽

Stem And Progenitor Cells

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Activation of OCT4 Enhances Ex Vivo Expansion of Phenotypically Defined and Functionally Engraftable Human Cord Blood Hematopoietic Stem and Progenitor Cells By Regulating HOXB4 Expression

Blood ◽

10.1182/blood.v124.21.4332.4332 ◽

2014 ◽

Vol 124 (21) ◽

pp. 4332-4332

Author(s):

Xinxin Huang ◽

Scott Cooper ◽

Hal E. Broxmeyer

Keyword(s):

Cord Blood ◽

Progenitor Cells ◽

Ex Vivo ◽

Fold Increase ◽

Lentiviral Vectors ◽

Ex Vivo Expansion ◽

Transcriptional Factor ◽

Human Cord Blood ◽

Hematopoietic Stem ◽

Cd34 Cells

Abstract Allogeneic hematopoietic cell transplantation (HCT) is well established as a clinical means to treat patients with hematologic disorders and cancer. Human cord blood (CB) is a viable source of hematopoietic stem cells (HSC) for transplantation. However, numbers of nucleated cells retrieved, as well as limited numbers of HSC/progenitor cells (HPC) present, during collection may be problematic for treatment of adult patients with single CB HCT. One means to address the problem of limiting numbers of HSC/HPC is to ex vivo expand these cells for potential clinical use. While progress has been made in this endeavor, there is still a clinically relevant need for additional means to ex vivo expansion of human HSC and HPC. OCT4, a transcriptional factor, plays an essential role in pluripotency and somatic cell reprogramming, however, the functions of OCT4 in HSC are largely unexplored. We hypothesized that OCT4 is involved in HSC function and expansion, and thus we first evaluated the effects of OAC1 (Oct4-activating compound 1) on ex vivo culture of CB CD34+ cells in the presence of a cocktail of cytokines (SCF, TPO and Flt3L) known to ex vivo expand human HSC. We found that CB CD34+ cells treated with OAC1 for 4 days showed a significant increase (2.8 fold increase, p<0.01) above that of cytokine cocktail in the numbers of rigorously defined HSC by phenotype (Lin-CD34+CD38-CD45RA-CD90+CD49f+) and in vivo repopulating capacity in both primary (3.1 fold increase, p<0.01) and secondary (1.9 fold increase, p<0.01) recipient NSG mice. OAC1 also significantly increased numbers of granulocyte/macrophage (CFU-GM, 2.7 fold increase, p<0.01), erythroid (BFU-E, 2.2 fold increase, p<0.01), and granulocyte, erythroid, macrophage, megakaryocyte (CFU-GEMM, 2.6 fold increase, p<0.01) progenitors above that of cytokine combinations as determined by colony assays. To further confirm the role of OCT4 in human HSC, we performed OCT4 overexpression in CB CD34+ cells using lentiviral vectors and found that overexpression of OCT4 also resulted in significant increase (2.6 fold increase, p<0.01) in the number of phenotypic HSC compared to control vectors. Together, our data indicate that activation of OCT4 by OAC1 or lentiviral vectors enhances ex vivo expansion of cytokine stimulated human CB HSC. HOXB4 is a homeobox transcriptional factor that appears to be an essential regulator of HSC self-renewal. Overexpression of HOXB4 results in high-level ex vivo HSC expansion. It is reported that OCT4 can bind to the promoter region of HOXB4 at the site of 2952 bp from the transcription start point. We hypothesized that activation of OCT4 might work through upregulation of HOXB4 expression to ex vivo expand HSC. We observed that the expression of HOXB4 was largely increased (2.3 fold increase, p<0.01) after culture of CB CD34+ cells with OAC1 compared to vehicle control. siRNA mediated inhibition of OCT4 resulted in the marked reduction of HOXB4 expression (p<0.01) in OAC1-treated cells indicating that OAC1 treatment lead to OCT4-mediated upregulation of HOXB4 expression in HSC. Consistently, siRNA-mediated knockdown of HOXB4 expression led to a significant reduction in the number of Lin-CD34+CD38-CD45RA-CD90+CD49f+ HSC in OAC1-treated cells (p<0.05), suggesting HOXB4 is essential for the generation of primitive HSC in OAC1-treated cells. Our study has identified the OCT4-HOXB4 axis in ex vivo expansion of human CB HSC and sheds light on the potential clinical application of using OAC1 treatment to enhance ex vivo expansion of cytokine stimulated human HSC. Disclosures Broxmeyer: CordUse: Membership on an entity's Board of Directors or advisory committees.

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The tetraspanin CD9 regulates migration, adhesion, and homing of human cord blood CD34+ hematopoietic stem and progenitor cells

Blood ◽

10.1182/blood-2010-04-281329 ◽

2011 ◽

Vol 117 (6) ◽

pp. 1840-1850 ◽

Cited By ~ 38

Author(s):

Kam Tong Leung ◽

Kathy Yuen Yee Chan ◽

Pak Cheung Ng ◽

Tze Kin Lau ◽

Wui Man Chiu ◽

...

Keyword(s):

Bone Marrow ◽

Cord Blood ◽

Progenitor Cells ◽

Critical Role ◽

Janus Kinase ◽

Human Cord Blood ◽

Hematopoietic Stem ◽

Short Term Exposure ◽

Cd34 Cells ◽

Cord Blood Cd34

Abstract The stromal cell–derived factor-1 (SDF-1)/chemokine C-X-C receptor 4 (CXCR4) axis plays a critical role in homing and engraftment of hematopoietic stem/progenitor cells (HSCs) during bone marrow transplantation. To investigate the transcriptional regulation provided by this axis, we performed the first differential transcriptome profiling of human cord blood CD34+ cells in response to short-term exposure to SDF-1 and identified a panel of genes with putative homing functions. We demonstrated that CD9, a member of the tetraspanin family of proteins, was expressed in CD34+CD38−/lo and CD34+CD38+ cells. CD9 levels were enhanced by SDF-1, which simultaneously down-regulated CXCR4 membrane expression. Using specific inhibitors and activators, we demonstrated that CD9 expression was modulated via CXCR4, G-protein, protein kinase C, phospholipase C, extracellular signal-regulated kinase, and Janus kinase 2 signals. Pretreatment of CD34+ cells with the anti-CD9 monoclonal antibody ALB6 significantly inhibited SDF-1–mediated transendothelial migration and calcium mobilization, whereas adhesion to fibronectin and endothelial cells was enhanced. Pretreatment of CD34+ cells with ALB6 significantly impaired their homing to bone marrow and spleen of sublethally irradiated NOD/SCID (nonobese diabetic/severe combined immune-deficient) mice. Sorted CD34+CD9− cells displayed lower bone marrow homing capacity compared with that of total CD34+ cells. CD9 expression on homed CD34+ cells was significantly up-regulated in vivo. Our results indicate that CD9 might possess specific functions in HSC homing.

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Liver Sinusoidal Endothelial Cells Promote the Expansion of Human Cord Blood Hematopoietic Stem and Progenitor Cells

International Journal of Molecular Sciences ◽

10.3390/ijms20081985 ◽

2019 ◽

Vol 20 (8) ◽

pp. 1985 ◽

Cited By ~ 3

Author(s):

Huilin Li ◽

Haiyun Pei ◽

Xiaoyan Xie ◽

Sihan Wang ◽

Yali Jia ◽

...

Keyword(s):

Endothelial Cells ◽

Cord Blood ◽

Progenitor Cells ◽

Ex Vivo ◽

Liver Sinusoidal Endothelial Cells ◽

Sinusoidal Endothelial Cells ◽

Differentiation Potential ◽

Hematopoietic Stem ◽

Stem And Progenitor Cells

Cord blood (CB) is an attractive source of hematopoietic stem cells (HSCs) for hematopoietic cell transplantation. However, its application remains limited due to the low number of HSCs/progenitors in a single CB unit and its notoriously difficulty in expanding ex vivo. Here, we demonstrated that the human fetal liver sinusoidal endothelial cells engineered to constitutively express the adenoviral E4orf1 gene (hFLSECs-E4orf1) is capable of efficient expansion ex vivo for human CB hematopoietic stem and progenitor cells (HSPCs). Coculture of CD34+ hCB cells with hFLSECs-E4orf1 resulted in generation of substantially more total nucleated cells, CD34+CD38− and CD34+ CD38−CD90+ HSPCs in comparison with that of cytokines alone after 14 days. The multilineage differentiation potential of the expanded hematopoietic cells in coculture condition, as assessed by in vitro colony formation, was also significantly heightened. The CD34+ hCB cells amplified on hFLSECs-E4orf1 were capable of engraftment in vivo. Furthermore, hFLSECs-E4orf1 highly expressed hematopoiesis related growth factor and Notch receptors. Accordingly, the CD34+ hCB cells amplified on hFLSECs-E4orf1 exhibited Notch signaling activation. Taken together, our findings indicated that FLSECs may potentially be the crucial component of the microenvironment to support recapitulation of embryonic HSC amplification in vitro and allow identification of new growth factors responsible for collective regulation of hematopoiesis.

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