Tailored Cytokine Optimization for ex vivo Culture Platforms Targeting the Expansion of Human Hematopoietic Stem/Progenitor Cells

Umbilical cord blood (UCB) has been established as an alternative source for hematopoietic stem/progenitor cells (HSPC) for cell and gene therapies. Limited cell yields of UCB units have been tackled with the development of cytokine-based ex vivo expansion platforms. To improve the effectiveness of these platforms, namely targeting clinical approval, in this study, we optimized the cytokine cocktails in two clinically relevant expansion platforms for HSPC, a liquid suspension culture system (CS_HSPC) and a co-culture system with bone marrow derived mesenchymal stromal cells (BM MSC) (CS_HSPC/MSC). Using a methodology based on experimental design, three different cytokines [stem cell factor (SCF), fms-like tyrosine kinase 3 ligand (Flt-3L), and thrombopoietin (TPO)] were studied in both systems during a 7-day culture under serum-free conditions. Proliferation and colony-forming unit assays, as well as immunophenotypic analysis were performed. Five experimental outputs [fold increase (FI) of total nucleated cells (FI TNC), FI of CD34+ cells, FI of erythroid burst-forming unit (BFU-E), FI of colony-forming unit granulocyte-monocyte (CFU-GM), and FI of multilineage colony-forming unit (CFU-Mix)] were followed as target outputs of the optimization model. The novel optimized cocktails determined herein comprised concentrations of 64, 61, and 80 ng/mL (CS_HSPC) and 90, 82, and 77 ng/mL (CS_HSPC/MSC) for SCF, Flt-3L, and TPO, respectively. After cytokine optimization, CS_HSPC and CS_HSPC/MSC were directly compared as platforms. CS_HSPC/MSC outperformed the feeder-free system in 6 of 8 tested experimental measures, displaying superior capability toward increasing the number of hematopoietic cells while maintaining the expression of HSPC markers (i.e., CD34+ and CD34+CD90+) and multilineage differentiation potential. A tailored approach toward optimization has made it possible to individually maximize cytokine contribution in both studied platforms. Consequently, cocktail optimization has successfully led to an increase in the expansion platform performance, while allowing a rational side-by-side comparison among different platforms and enhancing our knowledge on the impact of cytokine supplementation on the HSPC expansion process.

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Maintaining the self-renewal and differentiation potential of human CD34+ hematopoietic cells using a single genetic element

Blood ◽

10.1182/blood-2003-05-1762 ◽

2003 ◽

Vol 102 (13) ◽

pp. 4369-4376 ◽

Cited By ~ 87

Author(s):

James C. Mulloy ◽

Jorg Cammenga ◽

Francisco J. Berguido ◽

Kaida Wu ◽

Ping Zhou ◽

...

Keyword(s):

Progenitor Cells ◽

Ex Vivo ◽

Severe Combined Immunodeficiency ◽

Hematopoietic Cells ◽

The Self ◽

Differentiation Potential ◽

Self Renewal ◽

Hematopoietic Stem ◽

Human Cd34

AbstractHematopoiesis is a complex process involving hematopoietic stem cell (HSC) self-renewal and lineage commitment decisions that must continue throughout life. Establishing a reproducible technique that allows for the long-term ex vivo expansion of human HSCs and maintains self-renewal and multipotential differentiation will allow us to better understand these processes, and we report the ability of the leukemia-associated AML1-ETO fusion protein to establish such a system. AML1-ETO-transduced human CD34+ hematopoietic cells routinely proliferate in liquid culture for more than 7 months, remain cytokine dependent for survival and proliferation, and demonstrate self-renewal of immature cells that retain both lymphoid and myeloid potential in vitro. These cells continue to express the CD34 cell surface marker and have ongoing telomerase activity with maintenance of telomere ends, however they do not cause leukemia in nonobese diabetic-severe combined immunodeficiency (NOD/SCID) mice. Identification of the signaling pathways that are modulated by AML1-ETO and lead to the self-renewal of immature human progenitor cells may assist in identifying compounds that can efficiently expand human stem and progenitor cells ex vivo. (Blood. 2003; 102:4369-4376)

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Long-Term Production System of Red Blood Cells Using Human Cord Blood and Stromal Cells

Blood ◽

10.1182/blood.v116.21.3211.3211 ◽

2010 ◽

Vol 116 (21) ◽

pp. 3211-3211

Author(s):

Masayoshi Kobune ◽

Shohei Kikuchi ◽

Kazuyuki Murase ◽

Satoshi Iyama ◽

Tsutomu Sato ◽

...

Keyword(s):

Cord Blood ◽

Progenitor Cells ◽

Stromal Cells ◽

Production System ◽

Ex Vivo ◽

Erythroid Progenitor ◽

Differentiation Potential ◽

Hematopoietic Stem ◽

Human Stromal Cells

Abstract Abstract 3211 We have previously shown that primary human stromal cells and hTERT-transduced human stromal cells (hTERT-stromal cells) support cord blood (CB) hematopoietic stem/progenitor cells. However, it is unclear whether human stromal cells maintain the expansion of erythroid progenitor cells without losing erythroid differentiation potential for a long-term ex vivo culture. In an attempt to evaluate the efficacy of human stromal cells, erythroid induction was conducted by SCF, EPO and IGF-1, 2-week after expansion of CB CD34+ cells with or without human stromal cells. The maturation of erythroid cells were evaluated by morphological findings, transferrin receptor (TfR)/glycophorin A (GPA) expression and hemoglobin (Hb) synthesis (MCH, pg/cells). The number of BFU-E upon 2-week coculture with the hTERT-stromal cells was significantly higher than those without hTERT-stromal cells (BFU-E, 639±102 vs. 4078±1935, the initial cell number of BFU-E was 513±10). Hb concentration of erythroblasts that had been derived from coculture with stromal cells, was significantly higher than that derived from stroma-free condition 14 days after erythroid induction (MCH, 0.78±0.11 vs. 2.62±0.12; p<0.05). Moreover, cobblestone area (CA)-forming cells existed beneath stromal layer weekly produced the large number of BFU-E from 4th week to at least 8th week (the total number of BFU-E, 57246±1288)(Figure A). Notably, these BFU-Es derived from CA could simultaneously differentiate into orthophilic erythroblasts with nearly normal Hb synthesis (MHC, 24.5±6.4 pg/cell)(Figure B) and GPA expression. Furthermore, most of these erythroblasts derived from CA underwent enucleation spontaneously after further 7 days culture. Thus, using hTERT-stromal cells, the long-term ex vivo erythroid production could be attained from CB cells. These findings contribute to constructing long-term of ex vivo erythroid production system using human stromal cells. Disclosures: No relevant conflicts of interest to declare.

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A human stromal-based serum-free culture system supports the ex vivo expansion/maintenance of bone marrow and cord blood hematopoietic stem/progenitor cells

Experimental Hematology ◽

10.1016/j.exphem.2005.03.017 ◽

2005 ◽

Vol 33 (7) ◽

pp. 828-835 ◽

Cited By ~ 78

Author(s):

Cláudia Lobato da Silva ◽

Raquel Gonçalves ◽

Kirsten B. Crapnell ◽

Joaquim M.S. Cabral ◽

Esmail D. Zanjani ◽

...

Keyword(s):

Bone Marrow ◽

Cord Blood ◽

Progenitor Cells ◽

Culture System ◽

Ex Vivo ◽

Ex Vivo Expansion ◽

Hematopoietic Stem ◽

Serum Free

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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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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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Isoform-specific involvement of Brpf1 in expansion of adult hematopoietic stem and progenitor cells

Journal of Molecular Cell Biology ◽

10.1093/jmcb/mjz092 ◽

2019 ◽

Vol 12 (5) ◽

pp. 359-371

Author(s):

Qiuping He ◽

Mengzhi Hong ◽

Jincan He ◽

Weixin Chen ◽

Meng Zhao ◽

...

Keyword(s):

Histone Acetylation ◽

Progenitor Cells ◽

Ex Vivo ◽

Chromatin Accessibility ◽

Differentiation Potential ◽

Self Renewal ◽

Hematopoietic Stem ◽

Phd Finger ◽

Lineage Differentiation ◽

Stem And Progenitor Cells

Abstract Bromodomain-containing proteins are known readers of histone acetylation that regulate chromatin structure and transcription. Although the functions of bromodomain-containing proteins in development, homeostasis, and disease states have been well studied, their role in self-renewal of hematopoietic stem and progenitor cells (HSPCs) remains poorly understood. Here, we performed a chemical screen using nine bromodomain inhibitors and found that the bromodomain and PHD finger-containing protein 1 (Brpf1) inhibitor OF-1 enhanced the expansion of Lin−Sca-1+c-Kit+ HSPCs ex vivo without skewing their lineage differentiation potential. Importantly, our results also revealed distinct functions of Brpf1 isoforms in HSPCs. Brpf1b promoted the expansion of HSPCs. By contrast, Brpf1a is the most abundant isoform in adult HSPCs but enhanced HSPC quiescence and decreased the HSPC expansion. Furthermore, inhibition of Brpf1a by OF-1 promoted histone acetylation and chromatin accessibility leading to increased expression of self-renewal-related genes (e.g. Mn1). The phenotypes produced by OF-1 treatment can be rescued by suppression of Mn1 in HSPCs. Our findings demonstrate that this novel bromodomain inhibitor OF-1 can promote the clinical application of HSPCs in transplantation.

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Mimicking the Hematopoietic Niche Microenvironment Provides a Novel Strategy for Expansion of Hematopoietic and Megakaryocyte-Progenitor Cells from Cord Blood

Blood ◽

10.1182/blood.v112.11.3462.3462 ◽

2008 ◽

Vol 112 (11) ◽

pp. 3462-3462

Author(s):

Varda R Deutsch ◽

Einav Hubel ◽

Sigi Kay ◽

Tal Ohayon ◽

Ben-Zion Katz ◽

...

Keyword(s):

Cord Blood ◽

Progenitor Cells ◽

Mononuclear Cells ◽

Ex Vivo ◽

Myeloid Cell ◽

Fold Increase ◽

High Definition ◽

Short Term ◽

Hematopoietic Stem ◽

Hematopoietic Niche

Abstract While umbilical cord blood provides an important source of hematopoietic stem/progenitor cells (HSPC) for allogeneic transplantation in children, its use in adults is limited by the inadequate number of primitive stem cells and megakaryocyte progenitors (Mk-p) in single or even double CB units resulting in prolonged thrombocytopenia. Thrombopoietin treatment is not effective in these patients due to the paucity of target progenitors and patients require multiple platelet transfusions until the long-term engrafting cells can support thrombopoiesis, thus new modalities to increase progenitor cell dose are needed. A new transplantation strategy could involve the infusion of ex vivo-generated Mk-p together with unmanipulated single or double CB units. While CB CD34+ cells can be expanded by several reported methods, these rare cells cannot be sacrificed from the CB units due to their limited number. We propose a novel ex-vivo strategy to facilitate HSPC and Mk-p expansion from mononuclear cells (MNC) of a small aliquot of CB using conditions that mimic the hematopoietic niche, in short term cultures. Fibronectin (FN) was considered to be a prime candidate to support proliferation because it is a major extracellular matrix (ECM) component of all bone marrow hematopoietic microenvironments which is known to enhance viability and proliferation of HSPC. Other growth stimulators added were thrombopoietin (r-hu-TPO), the major physiological stimulator of MK and the synthetic hematopoietic stress peptide ARP derived from acetylcholinesterase, shown to increase transplantable Mk-p and produce human platelets in NOD/SCID mice (Pick et al, Blood 2006, Grisaru et al, J Imm 2006). High definition flow cytometry enabled assessing expansion of the SSClow/CD34high HSPC, and the SSClow/CD45dim/neg/CD41high Mk-p, and their subpopulations on day 0 and 10 of culture. True MK expansion was assessed by gating out of granulocyte and monocytes, which acquire CD41+ adherent platelets in culture. FN alone increased viability and expansion of HSPC by 6.9 fold and MK-p by 4-fold, while r-hu-TPO alone enhanced Mk-p proliferation with an average expansion of 8.3-fold in agreement with its known activity. Combining FN with r-hu-TPO produced a 25-fold increase in the number of MK-p while adding ARP to FN and r-hu-TPO was even more powerful, doubling the number of cells with a highly significant average expansion of 59-fold (p < 0.001). To define the progenitor subpopulations that contributed to Mk-p proliferation with FN, r-hu-TPO and ARP, we further analyzed the resulting subsets of MK-p cells, which also expressed either CD34, or the early myeloid marker CD33. The CD41high/CD34high population was increased by 4 fold, while the CD41high/CD33+ Mk-p, a subset with properties similar to clonogenic GEMM progenitors that could provide both myeloid and megakaryocytic cells post-transplant, were stimulated 30–50 fold. This notion is confirmed by the stimulation of CFU-MK and CFU-GEMM obtained under these conditions. Considering that expansion of MK-p requires proliferation of the HSPC precursor, we examined the proliferation of CD34+ progenitor cells and their subpopulations; CD34high/CD33+ or CD34high/CD41low uncommitted HSPC and CD41 high committed Mk subpopulations. The addition of FN alone stimulated CD34+ HSPC expansion by 6.9-fold (p < 0.05). All cultures that contained the ARP peptide maintained a high proliferation capacity, confirming that ARP protects and drives CD34+HSPC and early myeloid cell proliferation (Deutsch et al Exp Hem 2002). The addition of r-hu-TPO and ARP to FN produced a synergistic proliferative effect on the CD34+/CD41low HSPC stimulating a dramatic 440 fold increase of these uncommitted cells. These data support the notion that FN is protective and plays an essential role in enabling HSPC and MK-p expansion driven by r-hu-TPO and ARP. These conditions also supported MK maturation, as measured by increased high ploidy cells and elevated expression of GPIIb/IIIa detected by quantitative real time PCR. We demonstrate that expansion of both very early myeloid and Mk-p from a small fraction of the CB unit in short term cultures under conditions that mimic the hematopoietic niche is feasible, easy to perform and can comply with GTP requirements. This approach may lead to the development of more effective cell therapy modalities to facilitate myelopoiesis and platelet production following CBT.

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Decellularized Wharton jelly matrix: a biomimetic scaffold for ex vivo hematopoietic stem cell culture

Blood Advances ◽

10.1182/bloodadvances.2018019315 ◽

2019 ◽

Vol 3 (7) ◽

pp. 1011-1026 ◽

Cited By ~ 5

Author(s):

Dandan Li ◽

Grace Chiu ◽

Brea Lipe ◽

Richard A. Hopkins ◽

Jacquelyn Lillis ◽

...

Keyword(s):

Culture System ◽

Ex Vivo ◽

Cxcr4 Expression ◽

Differentiation Potential ◽

Self Renewal ◽

Hematopoietic Stem ◽

Megakaryocytic Differentiation ◽

Cd34 Cells ◽

Hematopoietic Niche

Abstract Hematopoietic stem progenitor cells (HSPCs) reside in the bone marrow (BM) hematopoietic “niche,” a special 3-dimensional (3D) microenvironment that regulates HSPC self-renewal and multipotency. In this study, we evaluated a novel 3D in vitro culture system that uses components of the BM hematopoietic niche to expand umbilical cord blood (UCB) CD34+ cells. We developed this model using decellularized Wharton jelly matrix (DWJM) as an extracellular matrix (ECM) scaffold and human BM mesenchymal stromal cells (MSCs) as supporting niche cells. To assess the efficacy of this model in expanding CD34+ cells, we analyzed UCB CD34+ cells, following culture in DWJM, for proliferation, viability, self-renewal, multilineage differentiation, and transmigration capability. We found that DWJM significantly expanded UCB HSPC subset. It promoted UCB CD34+ cell quiescence, while maintaining their viability, differentiation potential with megakaryocytic differentiation bias, and clonogenic capacity. DWJM induced an increase in the frequency of c-kit+ cells, a population with enhanced self-renewal ability, and in CXCR4 expression in CD34+ cells, which enhanced their transmigration capability. The presence of BM MSCs in DWJM, however, impaired UCB CD34+ cell transmigration and suppressed CXCR4 expression. Transcriptome analysis indicated that DWJM upregulates a set of genes that are specifically involved in megakaryocytic differentiation, cell mobility, and BM homing. Collectively, our results indicate that the DWJM-based 3D culture system is a novel in vitro model that supports the proliferation of UCB CD34+ cells with enhanced transmigration potential, while maintaining their differentiation potential. Our findings shed light on the interplay between DWJM and BM MSCs in supporting the ex vivo culture of human UCB CD34+ cells for use in clinical transplantation.

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Influence of the mesenchymal stromal cell source on the hematopoietic supportive capacity of umbilical cord blood-derived CD34+-enriched cells

Stem Cell Research & Therapy ◽

10.1186/s13287-021-02474-8 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Sara Bucar ◽

André Dargen de Matos Branco ◽

Márcia F. Mata ◽

João Coutinho Milhano ◽

Íris Caramalho ◽

...

Keyword(s):

Cord Blood ◽

Umbilical Cord ◽

Umbilical Cord Blood ◽

Mononuclear Cells ◽

Ex Vivo ◽

Fold Increase ◽

Cell Number ◽

Alternative Source ◽

Hematopoietic Stem ◽

Promising Alternative

Abstract Background Umbilical cord blood (UCB) is a clinically relevant alternative source of hematopoietic stem/progenitor cells (HSPC). To overcome the low cell number per UCB unit, ex vivo expansion of UCB HSPC in co-culture with mesenchymal stromal cells (MSC) has been established. Bone marrow (BM)-derived MSC have been the standard choice, but the use of MSC from alternative sources, less invasive and discardable, could ease clinical translation of an expanded CD34+ cell product. Here, we compare the capacity of BM-, umbilical cord matrix (UCM)-, and adipose tissue (AT)-derived MSC, expanded with/without xenogeneic components, to expand/maintain UCB CD34+-enriched cells ex vivo. Methods UCB CD34+-enriched cells were isolated from cryopreserved mononuclear cells and cultured for 7 days over an established feeder layer (FL) of BM-, UCM-, or AT-derived MSC, previously expanded using fetal bovine serum (FBS) or fibrinogen-depleted human platelet lysate (HPL) supplemented medium. UCB cells were cultured in serum-free medium supplemented with SCF/TPO/FLT3-L/bFGF. Fold increase in total nucleated cells (TNC) as well as immunophenotype and clonogenic potential (cobblestone area-forming cells and colony-forming unit assays) of the expanded hematopoietic cells were assessed. Results MSC from all sources effectively supported UCB HSPC expansion/maintenance ex vivo, with expansion factors (in TNC) superior to 50x, 70x, and 80x in UCM-, BM-, and AT-derived MSC co-cultures, respectively. Specifically, AT-derived MSC co-culture resulted in expanded cells with similar phenotypic profile compared to BM-derived MSC, but resulting in higher total cell numbers. Importantly, a subpopulation of more primitive cells (CD34+CD90+) was maintained in all co-cultures. In addition, the presence of a MSC FL was essential to maintain and expand a subpopulation of progenitor T cells (CD34+CD7+). The use of HPL to expand MSC prior to co-culture establishment did not influence the expansion potential of UCB cells. Conclusions AT represents a promising alternative to BM as a source of MSC for co-culture protocols to expand/maintain HSPC ex vivo. On the other hand, UCM-derived MSC demonstrated inferior hematopoietic supportive capacity compared to MSC from adult tissues. Despite HPL being considered an alternative to FBS for clinical-scale manufacturing of MSC, further studies are needed to determine its impact on the hematopoietic supportive capacity of these cells.

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