scholarly journals Cryopreservation of 13 Commercial Cannabis sativa Genotypes Using In Vitro Nodal Explants

Plants ◽  
2021 ◽  
Vol 10 (9) ◽  
pp. 1794 ◽  
Author(s):  
Cassandra D. Downey ◽  
Gregory Golenia ◽  
Ekaterina A. Boudko ◽  
Andrew Maxwell P. Jones

Cannabis has developed into a multi-billion-dollar industry that relies on clonal propagation of elite genetics with desirable agronomic and chemical phenotypes. While the goal of clonal propagation is to produce genetically uniform plants, somatic mutations can accumulate during growth and compromise long-term genetic fidelity. Cryopreservation is a process in which tissues are stored at cryogenic temperatures, halting cell division and metabolic processes to facilitate high fidelity germplasm preservation. In this study, a series of experiments were conducted to optimize various stages of cryopreservation and develop a protocol for long-term germplasm storage of Cannabis sativa. The resulting protocol uses a standard vitrification procedure to cryopreserve nodal explants from in vitro shoots as follows: nodes were cultured for 17 h in a pre-culture solution (PCS), followed by a 20-min treatment in a loading solution (LS), and a 60 min incubation in plant vitrification solution 2 (PVS2). The nodes were then flash frozen in liquid nitrogen, re-warmed in an unloading solution at 40 °C, and cultured on basal MS culture medium in the dark for 5 days followed by transfer to standard culture conditions. This protocol was tested across 13 genotypes to assess the genotypic variability. The protocol was successful across all 13 genotypes, but significant variation was observed in tissue survival (43.3–80%) and regrowth of shoots (26.7–66.7%). Plants grown from cryopreserved samples were morphologically and chemically similar to control plants for most major traits, but some differences were observed in the minor cannabinoid and terpene profiles. While further improvements are likely possible, this study provides a functional cryopreservation system that works across multiple commercial genotypes for long-term germplasm preservation.

Blood ◽  
2020 ◽  
Vol 136 (22) ◽  
pp. 2535-2547 ◽  
Author(s):  
W. Grey ◽  
R. Chauhan ◽  
M. Piganeau ◽  
H. Huerga Encabo ◽  
M. Garcia-Albornoz ◽  
...  

Abstract Expansion of human hematopoietic stem cells (HSCs) is a rapidly advancing field showing great promise for clinical applications. Recent evidence has implicated the nervous system and glial family ligands (GFLs) as potential drivers of hematopoietic survival and self-renewal in the bone marrow niche; how to apply this process to HSC maintenance and expansion has yet to be explored. We show a role for the GFL receptor, RET, at the cell surface of HSCs in mediating sustained cellular growth, resistance to stress, and improved cell survival throughout in vitro expansion. HSCs treated with the key RET ligand/coreceptor complex, glial-derived neurotrophic factor and its coreceptor, exhibit improved progenitor function at primary transplantation and improved long-term HSC function at secondary transplantation. Finally, we show that RET drives a multifaceted intracellular signaling pathway, including key signaling intermediates protein kinase B, extracellular signal-regulated kinase 1/2, NF-κB, and p53, responsible for a wide range of cellular and genetic responses that improve cell growth and survival under culture conditions.


2021 ◽  
pp. 185-190
Author(s):  
I.V. Mitrofanova ◽  
V.A. Brailko ◽  
N.P. Lesnikova-Sedoshenko ◽  
N.N. Ivanova ◽  
O.V. Mitrofanova

Blood ◽  
1996 ◽  
Vol 88 (11) ◽  
pp. 4149-4158 ◽  
Author(s):  
M Trevisan ◽  
XQ Yan ◽  
NN Iscove

Abstract This investigation was directed at separating long-term reconstituting (LTR) stem cells in normal murine marrow from hematopoietic precursors detectable in short-term assays in vitro and in vivo, and then at determining whether purified LTR cells could themselves form colonies in culture. To do so, it was first necessary to identify culture conditions that would induce their growth while preserving their long- term reconstituting capacity. Marrow was cultured with various cytokines in liquid suspension for 4 days, after which the surviving LTR activity was quantitated in a competitive in vivo assay. Activity was preserved near input levels with combined murine c-kit ligand (KL), interleukin-1 (IL-1), IL-6, and IL-11. When the cultures also included tritiated or unlabeled thymidine, LTR potential was eliminated, indicating that essentially all LTR cells were induced into cell cycle with these cytokines. To purify them, marrow was sorted on the basis of Ly6A expression and Rhodamine 123 retention. The Ly6AhiRh123ls fraction contained 85% of total recovered LTR activity but only 1% of the recovered cells measured by multilineage colony formation in spleens or in vitro. This fraction was cultured in methyl cellulose with KL, IL-1, IL-6, and IL-11 for 4 to 6 days, after which colonies were isolated and injected into mice. High levels of permanent reconstitution were achievable in sublethally irradiated W41/W41 mice after the injection of a single reconstituting unit, and limiting dilution analysis estimated the frequency of multilineage LTR at 1 in 11,200 unpurified adult marrow cells. In either lethally irradiated normal or sublethally irradiated W41/W41 mice, 1-year lymphomyeloid reconstitutions were obtained from 1 in 65 to 84 colonies of 2 to 16 dispersed cells, but not from larger colonies or those with clumped cells. The results establish that resting marrow LTR cells can be separated from almost all of the more advanced clonogenic cells that are still pluripotential, can be induced to cycle in culture by defined cytokines with preservation of their reconstituting potential, and can be manipulated and assayed efficiently at single-cell and colony levels.


Blood ◽  
1986 ◽  
Vol 68 (6) ◽  
pp. 1348-1354 ◽  
Author(s):  
A Johnson ◽  
K Dorshkind

Abstract Hemopoiesis in long-term bone marrow cultures (LTBMC) is dependent on adherent stromal cells that form an in vitro hemopoietic microenvironment. Myeloid bone marrow cultures (MBMC) are optimal for myelopoiesis, while lymphoid bone marrow cultures (LBMC) only support B lymphopoiesis. The experiments reported here have made a comparative analysis of the two cultures to determine whether the stromal cells that establish in vitro are restricted to the support of myelopoiesis or lymphopoiesis, respectively, and to examine how the different culture conditions affect stromal cell physiology. In order to facilitate this analysis, purified populations of MBMC and LBMC stroma were prepared by treating the LTBMC with the antibiotic mycophenolic acid; this results in the elimination of hemopoietic cells while retaining purified populations of functional stroma. Stromal cell cultures prepared and maintained under MBMC conditions secreted myeloid growth factors that stimulated the growth of granulocyte-macrophage colonies, while no such activity was detected from purified LBMC stromal cultures. However, this was not due to the inability of LBMC stroma to mediate this function. Transfer of LBMC stromal cultures to MBMC conditions resulted in an induction of myeloid growth factor secretion. When seeded under these conditions with stromal cell- depleted populations of hemopoietic cells, obtained by passing marrow through nylon wool columns, the LBMC stromal cells could support long- term myelopoiesis. Conversely, transfer of MBMC stroma to LBMC conditions resulted in a cessation of myeloid growth factor secretion; on seeding these cultures with nylon wool-passed marrow, B lymphopoiesis, but not myelopoiesis, initiated. These findings indicate that the stroma in the different LTBMC are not restricted in their hemopoietic support capacity but are sensitive to culture conditions in a manner that may affect the type of microenvironment formed.


2007 ◽  
Vol 74 (9) ◽  
pp. 1149-1156 ◽  
Author(s):  
Raúl Fernández-Gonzalez ◽  
Miguel Angel Ramirez ◽  
Ainhoa Bilbao ◽  
Fernando Rodríguez De Fonseca ◽  
Alfonso Gutiérrez-Adán

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 3546-3546
Author(s):  
Sawa Ito ◽  
A. John Barrett ◽  
Andre Larochelle ◽  
Nancy F. Hensel ◽  
Keyvan Keyvanfar ◽  
...  

Abstract Abstract 3546 Because MSC support the growth and the differentiation of normal hematopoietic stem cells we hypothesized that MSC might also support leukemia cells, in particular leukemia stem cells (LSC) in vitro. We cultured blast cells from patients with acute myelogenous leukemia (AML) in liquid medium to study persistence of stem-cell-like and differentiated leukemia cell populations by flow cytometry, with and without MSC and additional growth factors. Cryopresrerved peripheral blood mononuclear cells (PBMC) were obtained from 6 AML patients (mean Age 47, range 23–74). Leukemia blasts were isolated by sorting live (propidium iodide (PI)-negative) CD34+ lineage (CD2+, CD3+, CD14+ and CD19+) -negative cells using a FACS ARIA II cell sorter (BD). Sorted blasts (2.5 ×105 cells) were co-cultured with an equal number of irradiated MSC derived from healthy donor bone marrow in RPMI medium supplemented with 10% human serum, with or without a human cytokine (CYTO) mixture (50 ng/ml interleukin 3, 150 ng/ml stem cell factor, and 150ng/ml Flt-3 ligand). MSC were replenished every two weeks. The phenotype of cultured cells was analyzed weekly using fluorescently-conjugated monoclonal antibodies against CD34, CD38, and CD45, plus the lineage panel and a dead cell exclusion dye Cell cycle analysis with Hoeschst 33342 and Pyronin Y was performed on cells co-stained with CD34, CD45 and PI. Primary leukemia samples were phenotypically heterogeneous with respect to proportions of cells (co-)staining for CD34 and CD38 as previously reported: three samples showed CD34+CD38- predominance (LSC-like leukemia), and three were CD34+CD38+ (common myeloid progenitor (CMP)-like leukemia). LSC-like leukemia maintained viable CD34+CD38- cells for at least 6 weeks when co-cultured with MSC alone, in contrast to cultures with cytokines or medium only which showed rapid decline in the LSC populations and no prolonged maintenance of viable cells (p=0.0005) (Figure, left panel). CMP-like leukemia maintained their CD34+CD38+ phenotype when co-cultured with MSC alone but persistence of this subset was not significantly different from the other culture conditions (p=0.5) and no culture remained viable after 4 weeks (Figure, right panel). Cell cycle analysis showed that co-culture with MSC maintained CD34+ blasts in G0 significantly more than other culture conditions (P<0.0001). We conclude that MSC support the maintenance of a leukemia stem cell phenotype in a long- term (6 week) in vitro culture system. The differential capacity of MSC to support LSC- like and CMP- like leukemia may be associated with the different frequency of leukemia initiating cells within each leukemic blast population. NSG mice xenotranplant model experiments are ongoing to confirm this hypothesis. Co-culture of LSC with MSC represents a simple approach to maintain LSC in vitro and could be utilized to screen the drug targeting LSCs. Further study of the effect of MSC on LSC would elucidate a potential mechanism whereby the marrow microenvironment serves as a reservoir of persisting leukemia after remission induction chemotherapy. Disclosures: No relevant conflicts of interest to declare.


2015 ◽  
Vol 14 (1) ◽  
Author(s):  
Wanlapa Roobsoong ◽  
Chayada S Tharinjaroen ◽  
Nattawan Rachaphaew ◽  
Porpimon Chobson ◽  
Louis Schofield ◽  
...  

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