Synthetic mRNA reprogramming efficiency in human dermal fibroblasts

Answer

There is no single source-reported efficiency for synthetic modified-mRNA reprogramming of human dermal fibroblasts; the verified figures come from different cells, counting rules and denominators. The exact BJ neonatal foreskin rows of Warren 2010 report 2.0% without and 2.6% with Y-27632 at replating, scored at day 18 as TRA-1-60-positive colonies against initial cells plated scaled to the replated fraction e3ad92ec de46ef38 227f6749 f5f05e23. Schlaeger 2015 prints 2.1%, but as the mean of the 3 successfully reprogrammed samples with a 27% success rate 39fe0706 222931c6 2728f7b9 4270d4de e94074bb. The adult type 1 diabetes patient fibroblast line in Liu 2014 gave 0.044% (11 colonies) against 0.56% (56 colonies) for BJ, counted by morphology with no stated denominator; the record's raw_data.finding quotes the paper: 'The efficiency of iPSC derivation was therefore 0.56% and 0.044% in BJ and MMCF1 fibroblasts, respectively' 2f52b2cd. These are different experimental summaries, not estimates of one conversion probability.

Summary

Reported values run from 0.044% 2f52b2cd to 779.2% f89a7e9a. The 779.2% (about 800% in the text) is 3,896 +/- 131.14 TRA-1-60-positive colonies from 500 input cells in an mRNA-plus-miRNA-mimic protocol on neonatal FN2 fibroblasts; it exceeds 100% because one input cell can give several sister colonies, and it is not an adult dermal result f89a7e9a 0d9833f0. Percentages in this corpus count different things: split-adjusted colony yield, a success-conditioned geometric mean, a range, morphology-counted colonies, and colonies per input cell. The platform's own list of flagged measurement conflicts for this task is reproduced pair by pair in the table below; each pair is addressed there and none is a same-condition replication.

Raw counts and qualitative yields are kept as printed and not converted: 21 colonies from 150,000 seeded Lonza fibroblasts 7ce2b476, 5-10 colonies per well from about 50,000 BJ cells 3b618619, hundreds of colonies per well from 3,000 cells f426baed, and thousands from as few as 500 cells 96a844da.

Evidence

Warren 2010 (Cell Stem Cell): keep the exact Table 1 row

Denominator, from the Table 1 legend: average TRA-1-60-positive colonies per well divided by the initial number of cells plated, scaled to the fraction replated. Feeders are gamma-irradiated human neonatal fibroblasts at 33,000 cells/cm2; n=6 per condition unless stated.

| Row | Source-reported efficiency | Colonies/well | Conditions | Evidence | |---|---|---|---|---| | BJ KMOSL, Y27632 absent | 2.0% | 249 +/- 21 | 300,000 cells plated, split day 7, well fraction 1/24, scored day 18; oxygen not stated for this row | e3ad92ec de46ef38 | | BJ KMOSL, Y27632 present | 2.6% | 326 +/- 49 | same, ROCK inhibitor at passaging | 227f6749 f5f05e23 | | dH1f KMOSL, 20% O2 | 2.9% | 243 +/- 42 | ES-derived fibroblasts, 50,000 cells plated, split day 6, well fraction 1/6, scored day 18 | 2a84b21f | | dH1f KMOSL, 5% O2 | 4.4% | 367 +/- 38 | same block, 5% oxygen | 94d37e36 | | dH1f KMOS, 20% / 5% O2 | 0.6% / 2.7% | 48 +/- 18 / 228 +/- 30 | same block | 2a84b21f 94d37e36 | | dH1f KMOS RNA vs virus | 1.4% RNA (virus 0.04%) | 229 +/- 39, n=18 | 100,000 cells, split day 6, well fraction 1/6, 5% O2 for RNA | ee0af689 |

The repeated BJ and Schlaeger extractions describe the same experiments and are not independent replications. dH1f cells are human ES-derived fibroblasts, not a dermal biopsy line, so 2.9% and 4.4% are context, not dermal values. For the RNA-versus-virus row, the verified record quotes that the retroviral cultures were fixed on day 32 and both arms were then immunostained for TRA-1-60; another verified record and the task background place RNA scoring near day 18. The primary text does not settle the RNA fixation date, so it is left as unresolved and the BJ day-18 caption is not transferred to that row ee0af689 de46ef38.

Schlaeger 2015 (Nature Biotechnology)

2.1% is a geometric mean over successful samples only (3 of 11 tested, 27% success), 'emerging hiPSC colonies per starting cell number'; it is not an average over every attempt. Cells are neonatal BJ and patient skin fibroblasts; 50,000 target cells on day -1 onto NuFF feeders at 250,000 per well; 5% oxygen after the first transfection; transfections through day 16 222931c6 39fe0706 2728f7b9 4270d4de e94074bb. Scoring day and replating are not stated for the mRNA arm. Verified records attribute TRA-1-60 to this figure differently: two say the quoted TRA-1-60 sentence belongs to the Sendai method paragraph, two others record TRA-1-60 as its marker. The marker is therefore left as not separately stated for mRNA.

Other source-reported percentages and counts

| Study and cells | Figure as reported | Scoring and marker | Seeding, split, substrate, oxygen | Evidence | |---|---|---|---|---| | Liu 2014; adult T1D patient fibroblasts MMCF1 and neonatal BJ | 0.044% (11 colonies) and 0.56% (56 colonies); denominator not stated, seeding 2.5 x 10^4 (MMCF1) and 1 x 10^4 (BJ) cells/well given separately | Day 18 for MMCF1; BJ day not stated; hESC-like morphology, no TRA-1-60 | split and oxygen not stated; Stemgent kit, 18 days | 2f52b2cd | | Steinle 2019; neonatal NuFF | 0.8%, 20-25 colonies/well | day 19-21; marker defining the count not stated | 30,000 cells (Methods give 20,000) on NuFF feeders at 250,000 per well; 5% O2; split not stated | c5d23789 | | Durruthy-Durruthy 2014; BJ, HUF1, HUF58, GM13325 | 0.2-4% (range; 4 is its upper end) | TRA-1-60; count day and denominator not stated | 1x10^4-5x10^4 cells per six-well well, feeder-free on gelatin, 5% O2 | 3ce06467 | | Warren 2012; XFF xeno-free neonatal fibroblasts | about 250 colonies/well = 0.5% (CV 21%, n=3); L-Myc about 0.02%; 24-hour regime about 2% | day 15 (day 11 for the 24-hour regime), TRA-1-60 | 50K cells/well, CELLstart, feeder-free, no passaging, 5% O2 | 21b8d637 | | Kuebler 2026; fibroblast subset | mean 0.221%, SD 0.235, median 0.191% (all cell types mean 0.177%) | emerged around day 10; counting day and marker not fixed | 7.5x10^4 cells, Matrigel, 5% O2; non-modified RNAs plus immune-evasion RNA and miRNA | 101f4b50 | | Kogut 2018; neonatal FN2, adults F40, F62, F50, senescent F50S | 779.2 +/- 26.23% (3,896 colonies from 500 cells); F40 72.67 +/- 4.67% (2,000 cells/well), F62 60.93% (3,000), F50 36.43% (5,000), F50S 0.33% (100,000) | day 18, TRA-1-60 | feeder-free Geltrex, 5% O2, seven transfections every 48 h; with miRNA mimics | f89a7e9a 0d9833f0 | | Gaignerie 2018; Lonza fibroblasts | 21 colonies from 150,000 seeded cells; no percentage | count day and marker not stated | Matrigel, 20% O2, split not stated; modified nucleosides not established | 7ce2b476 | | Sridhar 2016; BJ | 5-10 colonies/well from about 50,000 cells; no percentage | within 3 weeks; live TRA-1-60 | feeder, oxygen, split not stated | 3b618619 | | McGrath 2018 (JoVE) | hundreds of colonies/well from 3,000 cells; no percentage | day 17-18, TRA-1-60 | laminin-521, 5% O2 | f426baed | | McGrath 2020 (chapter) | thousands of colonies from as few as 500 cells; qualitative | colonies by day 18-22; count day not specified | laminin-521, 5% O2 | 96a844da |

Kuebler's primary Methods describe non-modified RNAs, so it is a synthetic-RNA boundary case, not a strict modified-mRNA estimate. Kogut's regimen includes miRNA mimics and FN1/FN2 are described as primary neonatal fibroblasts, with 'dermal' not stated in the paper 0d9833f0.

Every platform-flagged measurement conflict

| Left | Right | Why this is not a same-condition conflict | |---|---|---| | 2.9% 2a84b21f | 0.5% 21b8d637 | ES-derived dH1f at 20% O2 with feeders and a day-6 split, day 18, versus unpassaged XFF on CELLstart at 5% O2, day 15; cell origin, matrix, cocktail and count day differ. | | 2.0% e3ad92ec | 4.0% 3ce06467 | Split-adjusted BJ row versus the upper end of a multi-line range with no stated denominator or count day. | | 2.0% e3ad92ec | 0.5% 21b8d637 | BJ with feeders and a day-7 split versus feeder-free unpassaged XFF; cocktail and scoring day differ. | | 2.6% f5f05e23 | 4.0% 3ce06467 | Y-27632-positive BJ row versus a range endpoint; cell composition, matrix and denominator differ or are unknown. | | 2.6% f5f05e23 | 0.5% 21b8d637 | Split BJ culture versus unpassaged XFF; origin, matrix, cocktail and count day differ. | | 4.4% 94d37e36 | 2.1% 39fe0706 | ES-derived dH1f optimized condition versus a success-only geometric mean across skin samples; cocktail, culture and scoring day are not matched. | | 4.4% 94d37e36 | 0.5% 21b8d637 | dH1f versus XFF; replating, feeders versus matrix and count day differ. | | 2.1% 39fe0706 | 4.0% 3ce06467 | Success-conditioned mean versus range endpoint; sample population, feeder environment and denominator are not matched. | | 2.1% 39fe0706 | 0.5% 21b8d637 | Sample selection, substrate, RNA regimen and endpoint differ. | | 2.0% de46ef38 | 4.0% 3ce06467 | The same BJ experiment as the 2.0% pair above, extracted again; not a second result. | | 2.0% de46ef38 | 0.5% 21b8d637 | The same BJ experiment extracted again; the XFF protocol and endpoint differences persist. | | 4.0% 3ce06467 | 0.5% 21b8d637 | Upper end of a multi-line range versus one XFF condition; denominator, cells, culture and count day are not matched. |

No pair shares cell type, delivery protocol, scoring day, marker and denominator, so the corpus does not settle whether any residual difference is biological. No cross-paper ratio or pooled efficiency is computed. Liu 2014 adds a pair the platform did not flag: 0.044% in MMCF1 against 0.56% in BJ inside one paper, with each line seeded at a different density and neither denominator stated, so the gap cannot be assigned to adult versus neonatal origin 2f52b2cd.

Methodology

Every verified fact supplied for this task is used and cited above. Repeated Warren and Schlaeger extractions are grouped by experimental row, not counted as new samples. Percentages, raw colony counts, success-conditioned means, ranges and qualitative yields are kept as separate quantities; nothing was converted, no denominator was reconstructed from seeding numbers, and no cross-paper ratio was formed. Where records attribute the same detail differently (Schlaeger's marker, Warren's RNA-arm scoring day, Steinle's seeding number) both readings are reported and the field is left unresolved.

Limitations

• The corpus cannot give a universal efficiency for adult dermal fibroblasts under one modified-mRNA protocol: several headline rows are neonatal e3ad92ec c5d23789, one is ES-derived 94d37e36, one is non-modified RNA 101f4b50, and some include miRNA f89a7e9a.
• Numerator, endpoint and denominator are often not jointly stated 3ce06467 2f52b2cd 7ce2b476 96a844da 21b8d637.
• Steinle's printed 0.8% does not follow simply from 20-25 colonies and the stated seeding number, and the paper cautions that colonies may derive from daughter cells c5d23789.
• Warren's RNA-arm scoring date and Schlaeger's mRNA-arm marker are unresolved in the extractions ee0af689 2728f7b9 4270d4de e94074bb 222931c6.