UM171

Hematopoietic cytokines; human hematopoietic stem cell self-renewal

NSG mice were injected with CD34+ CB cells that had been originally cultured in DMSO or UM171. Levels of human cell engraftment were determined for ~300 mice and represented in the form of a heat map. Analysis of this dataset indicates two emerging patterns of human reconstitution, one from predominantly lymphomyeloid LT-HSCs, observed at high cell doses with most conditions, and the other from LT-HSCs that display a lymphoid-deficient differentiation phenotype mostly observed with UM171 treatment. Neither B lymphopoiesis nor the frequency or number of lymphomyeloid LT-HSCs is negatively affected by UM171. The impact of UM171 on LT-HSC was preserved at 30 weeks posttransplantation, at which time multilineage contribution remained obvious at the high cell dose.

Primary screen and flow cytometry [1]
CD34+ mPB cells were seeded at 2000 cells per well in the presence of chemical compounds. Relative and absolute numbers of CD34+CD45RA- cells were determined after 7 days of culture using flow cytometry. Primary hits were selected as indicated in Fig. 1. The primary screen was performed with a LSRII flow cytometer equipped with a high throughput screening module. Cells were stained in PBS supplemented with 2% fetal bovine serum (FBS) at 4°C for 15 minutes with APC-labelled anti-human CD34 and PE-labelled anti-human CD45RA.
Cell phenotypes in fresh and expanded cells were measured using a combination of the following antibodies and fluorophores: FITC-labelled anti-human CD34, PE-labelled anti-human CD45RA, PE-Cy7-labelled antihuman CD38, APC-labelled anti-human CD90 (BioLegend), PE-Cy5- labelled anti-human CD49f and APC-labelled anti-human EPCR (eBioscience). Stained cells were washed once with PBS supplemented with 2% FBS and analyzed.

Transplantation and monitoring of human CD34+ CB cells in NSG mice [1]
All experiments with animals were conducted under protocols approved by the Animal Care Committee of Université de Montréal. Fresh CD34+ CB cells or their progeny present in 12-day cultures were transplanted by tail vein injection into sub-lethally irradiated (250 cGy, <24 hr before transplantation) 8 to 12 week-old female NSG (NODScid IL2R􀜵null, Jackson Laboratory). Human cells in NSG bone marrow (BM) was monitored by flow cytometry 20 and 30 weeks post-transplantation. NSG BM cells were collected by femoral aspiration (at week 20) or by flushing the two femurs, tibias and hips when animals were sacrificed at week 30. For secondary transplants, 25,000 freshly isolated (uncultured) CD34+ CB cells or the progeny of the equivalent of 10,000 freshly isolated CD34+ CB cells that had been originally cultured in DMSO or UM171 were injected into secondary sub-lethally irradiated NSG mice. BM cells of the secondary mice were harvested and analyzed 18 weeks post-transplantation. Flow cytometry analysis was performed on freshly collected BM cells. Cells were treated with 1x red blood cell lysis buffer, washed and stained with pacific blue-labelled anti-human CD45, APC-eFluo-labelled 780 anti-mouse CD45, PE-labelled anti-human CD33, PE-Cy5 labelled antihuman CD11b, FITC-labelled anti-human CD15, APC-Cy7 labelled anti-human CD14, PE-Cy7 labelled anti-human CD19, FITC-labelled anti-human CD3, APC-labelled antihuman CD71, PE-labelled anti-human glycophorin A (GPA), FITC-labelled anti-human CD41. Cells then were washed and analyzed using a FACSCanto II. BD FACSDiva or FlowJo software were used to analyze the flow cytometry data. The correspondence between antibody labeling and cell populations is as follows: CD11b, CD14, CD15, CD33 (monocytes and granulocytes); CD56 (NK cells); CD71, GPA, CD41 (erythroid and megakaryocyte lineages); CD19, CD3 (B and T cells) and CD34 (stem/progenitor cells).
A total of 300,000 BM cells were analyzed per mouse. Considering current FACS limit of detection of 1 per 20,000 events, a minimum threshold of 15 human cells could be detected in these mice. Because some lymphoid cells are long-lived, their presence after a long time can be potentially misleading for assessing LT-HSC activity, which is more conservatively assessed by relying on detecting the sustained output of short-lived cells (e.g., granulocytes).To detect human LT-HSC contribution to NSG reconstitution, we arbitrarily set the lower limit of human cell engraftment at 10-times the FACS threshold or 150 myeloid cells per 300,000 BM cells. T-cell reconstitution was not monitored because it correlates poorly with LT-HSC engraftment in other studies (5) and may represent graft versus host disease, a state that could be associated with peripheral amplification of T-cells not reflecting LT-HSC activity. Similarly, low levels of B-cell restricted reconstitution (<0.05%) was not considered for LT-HSC evaluation because of the extended longevity of B lymphocytes in vivo. However, these criteria led to the reclassification of only 5 mice in our cohort of 300.

[1]. Science.2014 Sep 19;345(6203):1509-12.

C25H27N9

453.54

453.2389419

1448724-09-1

Off-white to yellow solid

4.5

123Ų

CN1N=C(C2=CC3=C(C=C2)C4=C(N[C@H]5CC[C@@H](CC5)N)N=C(CC6=CC=CC=C6)NC4=N3)N=N1

AZXXGVPWWKWGAE-IYARVYRRSA-N

InChI=1S/C25H27N9/c1-34-32-23(31-33-34)16-7-12-19-20(14-16)28-25-22(19)24(27-18-10-8-17(26)9-11-18)29-21(30-25)13-15-5-3-2-4-6-15/h2-7,12,14,17-18H,8-11,13,26H2,1H3,(H2,27,28,29,30)/t17-,18-

(1r,4r)-N1-(2-Benzyl-7-(2-methyl-2H-tetrazol-5-yl)-9H-pyrimido[4,5-b]indol-4-yl)cyclohexane-1,4-diamine

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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 (Please use freshly prepared in vivo formulations for optimal results.)