Abstract

Somatic cell nuclear transfer extensively reprograms the transferred nucleus, but the process is neither instantaneous nor complete. Whether an acquired state of the nuclear donor can influence behavioral development in a resulting clone is unknown. We examined that possibility through a retrospective macaque observation, a prospective mouse experiment using paired pretraining and posttraining donor cells, two human archival investigations, and exploratory molecular analysis.

A cloned macaque developed a stable, unnecessary object-correction sequence corresponding to an action acquired by its nuclear donor during a documented period of apparatus malfunction. We subsequently trained eight mouse donors to perform an assigned leftward or rightward return during acquisition of a fixed-ratio lever-press task. Fibroblasts were collected before training and again after the assigned movement had either persisted or ceased following removal of the guide that originally required it. Four additional donors provided time-separated handled-control samples. Clones were reared without exposure to the donor task and tested under blinded conditions.

Nine of twenty-one clones derived from posttraining cells developed a stable additional turn after ordinary operant shaping, and seven turned in the direction assigned to the associated donor. Stable turns occurred in two of nineteen pretraining clones, one of which matched the donor assignment, and in one of twelve handled-control clones. Posttraining clones did not reproduce the donor task before shaping, learn it faster, or perform it more efficiently. Expression of the additional movement decreased when the chamber’s spatial organization was changed.

The human component began with iPSCs-11, whose redundant receiver-handling sequence was documented before the nuclear donor’s identity and occupational history were released. The donor had acquired the same sequence during an intermittent hook-switch failure and retained it after the instrument was replaced. A subsequent criterion-based archival review retained one additional case, GIAM-27, whose English pronunciation was placed by blinded reviewers within a Rhine Franconian range compatible with the donor’s West Palatine background. I regard this second correspondence as substantially less specific because shared anatomy, abnormal development, incomplete exposure history, and archival selection remain serious alternatives. Neither case demonstrated transferred object knowledge, language, declarative memory, or personal recollection.

Methylation profiling showed variable retention of donor-cell state after nuclear transfer and a weak, incomplete association with behavioral expression in motor cortex and dorsal striatum. No molecular feature predicted an individual clone’s behavior or specified turn direction. We use behavioral residue for the partial, cue-dependent recurrence of an acquired component of donor behavior without recovery of the knowledge or circumstances that originally made that component useful. The findings support the possibility that donor history can sometimes alter later action organization after nuclear transfer. They do not identify the causal substrate or explain how any retained donor-cell state could preserve the historical specificity of an acquired action.

Introduction

Somatic cell nuclear transfer requires a differentiated nucleus to support development for a second time. The transferred nucleus must be reorganized from a state established in another tissue, another body, and at another developmental age. That reorganization can produce a complete animal, but it does not restore every molecular feature uniformly to an embryonic condition. Persistent histone methylation and abnormal DNA re-methylation remain barriers to normal development after nuclear transfer,[1,2] and the production of live cloned macaques has shown both that primate SCNT is possible and that outcome still depends partly on the source and condition of the donor cell.[3]

Most consequences of incomplete reprogramming have been studied through survival, placental development, anatomy, organ function, and gross developmental abnormality. Behavior presents a less convenient problem. A clone can remain entirely within the expected species repertoire while differing in the timing, organization, or persistence of one action. Donor and clone also share a nuclear genome and may therefore share body structure, sensory biases, motor constraints, temperament, and learning tendencies. Behavioral resemblance is expected in many domains. By itself, it is not evidence that anything acquired by the donor survived nuclear transfer.

The relevant question is narrower and historical. If a donor develops an action because of a particular event, and a clone later reproduces an unnecessary component of that action without documented exposure to the event or to the donor’s behavior, ordinary genetic similarity becomes a less complete explanation. Complexity is not the issue. A simple movement can be informative when its presence depends on an identifiable history; an elaborate behavior can be uninformative when it is species-typical or easily learned from the current environment.

This study began with such a distinction.

SCNT-M4, a cloned cynomolgus macaque in a longitudinal developmental program, repeatedly displaced a handled object to the left after contact with an insertion opening, withdrew it, and initiated a second approach. The sequence did not improve insertion and persisted after direct insertion had been learned. Lateral corrections themselves were not unusual, and several control animals made them during early acquisition. What distinguished the SCNT-M4 sequence was its stable order and its persistence after the correction had ceased to be useful.

The associated donor archive was opened only after the clone sequence had been defined. The donor was found to perform the same leftward contact, withdrawal, and second approach. More importantly, the surviving record placed stable acquisition of that movement during a period of intermittent guide misalignment documented in the apparatus-maintenance log. The donor continued to use the correction after repair.

I considered the chronology more informative than the visual similarity. Two macaques can make similar movements for many reasons. It is harder to dismiss the history of an unnecessary movement when one record shows when and why that movement became useful. Even so, the observation remained retrospective. Donor and clone were studied within the same institution, some personnel worked across both periods, and the original apparatus was no longer available for inspection. We found no documented demonstration of the donor sequence to SCNT-M4 and no routine use of the archived footage during clone training. Yes, I checked. That does not mean every informal description or undocumented exposure can be excluded.

The macaque event therefore gave us a question, not an answer. We designed a prospective mouse experiment to separate donor genotype from donor training history. Fibroblasts were collected from the same donors before and after acquisition of an experimentally assigned directional action. During operant training, each donor was required to return to the lever by a leftward or rightward path. The guide imposing that route was later removed, allowing us to determine whether the additional turn persisted when it was no longer necessary. Clones derived from the paired samples were reared without the donor task and tested without guide panels.

The relevant outcome was not lever pressing or acquisition of a fixed-ratio schedule. Those were ordinary learned behaviors. The question was whether an unnecessary directional component associated with the donor’s training history would recur preferentially in clones derived from cells collected after that history had been established. An intact inherited task would have been a very different finding. The posttraining clones did not enter an unfamiliar chamber and spontaneously perform the donor’s five-press sequence. They required shaping, learned within the ordinary range, and adjusted when reinforcement conditions changed. The anomalous result concerned how an additional movement became organized after learning had taken place.

The human records entered the study differently and are not prospective replications. In iPSCs-11, a disconnected telephone receiver was lifted, fully replaced, immediately lifted again, and then placed against the head. The sequence was entered into the subject record, countersigned, and confirmed from the complete recording before the donor’s identity, occupation, or telephone history was released. The donor was subsequently found to have acquired the same redundant sequence during an intermittent hook-switch failure, retained it after the faulty instrument was replaced, and performed it before collection of the somatic cells later used for nuclear transfer.

No general human screening criterion existed before iPSCs-11 was noticed. Criteria for the remaining archival review were fixed only after the index sequence had been documented. I do not describe iPSCs-11 as the product of a blinded search through an unselected archive, because it was not one. Its evidentiary value depends instead on the order of documentation: the subject sequence was defined before the donor history capable of making that sequence interesting was known.

Five human subjects had records adequate for retrospective comparison. One additional case, GIAM-27, was retained. Her English pronunciation had attracted repeated clinical comment before the present review, and two blinded phonetic reviewers independently placed the surviving speech within a Rhine Franconian range. The nuclear donor had acquired West Palatine German near Kaiserslautern and retained a comparable regional influence in English.

This is an intriguing correspondence. It is also a much less convenient one. GIAM-27 and the donor shared a nuclear genotype; the subject underwent accelerated growth, uneven neurological development, progressive metabolic disease, and abnormal motor decline; detailed vocal-tract biomechanics were never studied; and continuous recording of her auditory environment did not exist. Several identified features can arise outside German speech, and neighboring regional varieties overlap. I therefore retain GIAM-27 as a boundary observation rather than treating it as equivalent to the telephone sequence or the prospective mouse result.

Neither human case suggests recovery of the donor’s language, autobiographical memory, or understanding of an object. iPSCs-11 did not dial, select numbers, wait for a response, or use the telephone communicatively. GIAM-27 produced no German vocabulary, grammar, or demonstrated comprehension. The macaque reproduced no knowledge of the donor’s apparatus history, and the mice did not know the donor task before training. Where recurrence occurred, it was narrower: an acquired component of action appeared again without the information that had originally given it a purpose.

We use donor-concordant procedural pattern for an acquired component of donor behavior that appears in a clone without documented exposure to the donor’s learning history. The term does not imply that a memory was stored in a fibroblast, that a neural representation survived transfer, or that the clone possesses the donor’s intention or recollection. We use behavioral residue for the broader phenomenon suggested here: partial, cue-dependent recurrence of such a component after nuclear transfer.

There are precedents for acquired physiological states influencing later development, including behavioral and physiological effects associated with parental conditioning, early stress, and sperm RNA.[4,5] Those studies do not demonstrate transmission of a specific learned action, and their biological routes are not equivalent to SCNT. They do establish the more limited point that experience can alter biological material in ways that matter later.

Epigenetic persistence is therefore an obvious candidate. Reprogrammed cells can retain molecular features associated with their previous state, and those features can influence differentiation.[6] Yes, this makes incomplete reprogramming relevant. It does not solve the present problem. A fibroblast nucleus does not contain the donor’s motor circuit, and persistence of a methylation difference does not explain how a later nervous system could preferentially reproduce the direction, order, or historically contingent structure of an acquired movement.

We therefore separated the behavioral question from the molecular one. The prospective mouse experiment tested whether donor training history altered later behavioral probability. Molecular analyses began only after behavioral classification and were exploratory. The study asks whether an acquired and historically identifiable component recurs more often from posttraining than pretraining cells; whether archival primate and human correspondences concern procedural organization rather than recovered knowledge; and whether incomplete reprogramming can provide a developmental route without being mistaken for an information-storage mechanism.

The evidence is not symmetrical. It supports the prospective behavioral comparison more strongly than the archival cases, and the archival cases more strongly than any molecular account. A behavioral effect can be real before its mechanism is known. A molecular association can also be real without explaining the specificity of the behavior. I have tried not to convert either uncertainty into a stronger claim merely because the stronger claim would be more satisfying.

Prospective Mouse Testing Separated Donor Genotype From Training History

Eight adult male donors entered operant training, and four additional donors served as handled controls. Skin fibroblasts were collected before chamber exposure and again after the corresponding training or control interval. The principal comparison therefore used cells from the same trained donor before and after acquisition of an experimentally assigned action, holding nuclear genotype constant within each pair.

Leftward or rightward return was assigned by block randomization before operant acquisition. Four donors were assigned to each direction, and personnel conducting later clone testing did not receive the allocation record. Donors learned a fixed-ratio-five lever-press task while a removable guide constrained the route from the reward port back to the lever. After stable task performance, the guide was removed and training continued in an unobstructed chamber.

The behavior of interest was not lever pressing. Every trained donor was expected to learn that. It was the additional directional return that remained after the guide no longer required it. Five donors continued to make the assigned turn before most new press sequences, two retained it intermittently, and one ceased within three sessions. Posttraining biopsies were collected only after persistence or cessation had been documented.

Handled donors entered the same type of chamber on a yoked schedule and received the same liquid reward, but the lever was unavailable and no directional guide was installed. This controlled the biopsy interval, repeated handling, chamber exposure, and reward consumption. It did not reproduce sustained lever pressing, route acquisition, or every physiological consequence of training. Yes, those omissions matter. We used the handled group for what it actually controlled and not as a substitute for the paired pretraining samples.

Nuclear transfer produced nineteen behaviorally eligible clones from pretraining cells, twenty-one from posttraining cells, and twelve from the second samples of handled-control donors. Developmental yield differed among lines. Clones were reared without operant chambers, guide panels, or donor training material, and personnel responsible for rearing, testing, and initial video coding did not know donor identity, collection period, or assigned direction.

Posttraining Clones Did Not Spontaneously Know the Donor Task

Before shaping, every clone received an unrewarded exposure to the unobstructed chamber. Lever contact was infrequent across groups. No clone produced a stable five-press sequence or repeatedly reproduced the associated donor turn. Posttraining clones did not approach the lever earlier, contact the reward port more frequently, or otherwise anticipate the reinforcement procedure.

This negative result matters. If the posttraining clones had entered the chamber already performing the donor sequence, I would describe the phenomenon differently. They did not. All animals underwent ordinary magazine training, lever shaping, and progression to the fixed-ratio-five schedule. Guide panels were never installed during clone training.

Stable Additional Turns Were More Frequent After Transfer From Posttraining Cells

A stable turn was defined before unblinding as the same directional movement occurring before at least two thirds of completed ratios in each of three consecutive sessions.

Nine of twenty-one posttraining clones met that criterion. Seven turned in the direction assigned to the associated donor, one turned in the opposite direction, and one retained an additional turn while alternating direction. Two of nineteen pretraining clones developed a stable turn; one matched the direction later assigned to its donor and one did not. One of twelve handled-control clones also developed a stable turn, but handled donors had no assigned direction and therefore no donor-matched classification.

Stable turning without regard to direction occurred in 9/21 posttraining clones, compared with 2/19 pretraining clones and 1/12 handled-control clones. In unclustered animal-level comparisons, posttraining differed from pretraining (odds ratio, 6.38; P = .034) and approached the same threshold against handled controls (odds ratio, 8.25; P = .054). These were secondary comparisons. A stable turn that does not match donor direction is not the primary outcome, and counting every clone as independent ignores the design.

The prespecified directional outcome occurred in 7/21 posttraining clones and 1/19 paired pretraining clones. At the animal level, the risk difference was 28.1 percentage points (95% CI, 2.9 to 49.8), the odds ratio was 9.00, and the two-sided Fisher exact P value was .046.

Clones from the same donor and fibroblast culture nevertheless shared genotype, cell preparation, production history, and other conditions. We therefore examined the primary outcome by donor line. At least one donor-matched stable turn occurred among posttraining clones from five of eight trained donor lines and among pretraining clones from one of the corresponding lines. The paired table contained one line positive in both conditions, four positive only after training, none positive only before training, and three positive in neither. The exact McNemar comparison favored posttraining cells but remained imprecise (5/8 versus 1/8 lines; P = .125).

Yes, the animal-level comparison crossed .05 and the donor-line comparison did not. Those statements are not contradictory. The first estimates expression among the clones produced in this series. The second asks whether the effect recurred across independently trained source animals and exposes how little independent replication an eight-donor SCNT experiment can provide. I regard the line-level analysis as the more conservative representation of the design.

Concordance was related imperfectly to persistence of the donor action. Four of five donors whose assigned turns remained persistent produced at least one matching posttraining clone. One of two donors with intermittent persistence did so. The donor whose turn ceased produced none. The ordering is suggestive, but it is not a dose-response result: one donor with persistent turning and one with intermittent turning produced no matching clone, and no line produced a matching movement in every clone.

Donor training history altered a distribution. It did not impose a behavioral fate.

Ordinary Task Learning Was Not Improved in Posttraining Clones

The median number of sessions required to reach stable fixed-ratio-five performance was 5 in the pretraining group (interquartile range, 4–7), 5 in the posttraining group (4–6), and 6 in the handled-control group (4–7). A time-to-criterion model provided no evidence of a source-cell group effect (P = .68).

Mean rewards per completed stable-performance session were 38.4 ± 8.1, 39.6 ± 7.7, and 37.9 ± 8.5 in the pretraining, posttraining, and handled-control groups, respectively. Incorrect reward-port approaches averaged 3.1 ± 1.4, 2.9 ± 1.3, and 3.2 ± 1.5. Source-cell group was not associated with reward yield (P = .74) or incorrect port approach (P = .81), and group-by-session interactions were not supported for these measures (all P > .20).

The values varied among donor lines, as expected in an F2 hybrid population and uneven production series. What they did not show was a consistent posttraining advantage. The additional turn was dissociable from competence at the task.

After stable classification, the fixed-ratio schedule was replaced with a variable-ratio schedule having the same mean response requirement. Six of the nine posttraining clones with stable turns continued to perform the additional movement after the constant grouping of five responses had been removed, while adjusting their reward-port behavior to the new schedule. The movement was therefore associated more closely with organization of the return to the lever than with knowledge of a five-response rule.

Expression Decreased When the Familiar Spatial Configuration Was Removed

The nine posttraining clones with stable turns were subsequently tested in a chamber with a different lever housing and reward-port arrangement. Four continued to turn consistently. Three showed the movement during the first session and then reduced or lost it. Two stopped.

The manipulation changed several visual and spatial features simultaneously, so it cannot identify a single eliciting cue. I do not claim that it does. The useful result is that expression was not equally stable across configurations.

The prospective experiment therefore produced donor-direction correspondence more often among clones derived from posttraining than paired pretraining cells. It did not produce inherited lever pressing, inherited reward knowledge, or spontaneous performance of the donor task. The most conservative description is that donor training history altered the probability with which an unnecessary directional component became organized during later learning.

That is already an unusual result. It does not need to be made larger than it is.

Molecular Profiling Identified Incomplete Donor-State Retention but No Representation of the Learned Action

Frozen aliquots of paired pretraining and posttraining fibroblasts remained available from all eight trained donors, together with time-separated samples from the four handled controls. We also retained SCNT blastocysts not transferred for gestation and terminal tissues from twenty-four behaviorally classified clones. The molecular study began after behavioral classification, and the clone subset was deliberately selected to represent every trained donor line, all seven posttraining animals with a stable donor-matched turn, atypical stable movements, and animals without a stable turn. This made the analysis useful for rare categories and post hoc by design. I do not treat it as an independent prospective test.

Genome-wide genotyping confirmed the expected nuclear relationships. We found no sample interchange or acquired sequence variant separating behavioral categories. Oocyte donors were distributed across pretraining and posttraining production, no mitochondrial haplotype was confined to donor-matched animals, and large acquired copy-number changes provided no obvious group separation. These findings do not make genotype irrelevant to individual variability, but they exclude several simpler explanations for a systematic difference between the paired cell periods.

Genome-wide methylation profiling identified differences between pretraining and posttraining fibroblasts in every trained donor. The number of qualifying differentially methylated regions ranged from 190 to 910 per donor, with a median of 430. Time-separated handled-control samples contained 70 to 300, with a median of 150. No region changed in the same direction in all eight trained pairs.

Posttraining was therefore not synonymous with changed because the donor learned the turn. The second biopsy also followed time, handling, chamber exposure, altered activity, reward consumption, and physiological change. Training may have contributed to donor-cell state; the paired samples cannot assign every molecular difference specifically to learning.

Most donor-state differences were no longer detectable after nuclear transfer. A small, variable fraction remained directionally associated with source-cell state in SCNT blastocysts, and the retained fraction overlapped time-associated retention in handled controls. No blastocyst pattern distinguished left-trained from right-trained donors.

That result deserves emphasis. We found evidence that some donor-cell state could survive reprogramming. We did not find a molecular sign saying left.

For adult tissues, retention was summarized as the proportion of source-cell-associated regions remaining shifted in the corresponding direction after adjustment for tissue and sequencing batch. The adult set comprised twenty-four clones: twenty-one posttraining animals—seven with donor-matched stable turns, one with an opposite turn, one with an alternating movement, and twelve without a stable turn—and three pretraining reference animals. The principal expression analysis compared the seven donor-matched posttraining clones with the twelve posttraining clones without a stable turn. The two posttraining animals with atypical stable movements and all three pretraining references were displayed separately.

In motor cortex, the seven donor-matched clones retained a median of 7.4% of the posttraining-associated donor state (interquartile range, 5.8%–9.1%), compared with 4.9% (3.6%–6.8%) among the twelve posttraining clones without a stable turn. The estimated median difference was 2.3 percentage points, with a nominal animal-level P value of .036. In dorsal striatum, the corresponding medians were 6.9% (5.3%–8.4%) and 4.7% (3.4%–6.3%), an estimated difference of 1.9 percentage points with nominal P = .044. No comparable separation appeared in cerebellum (4.6% versus 4.3%; P = .71) or quadriceps (3.8% versus 4.0%; P = .82).

After Benjamini–Hochberg correction across the four tissues, motor cortex and dorsal striatum each had q = .088. The distributions overlapped substantially. Some donor-matched animals fell within the nonexpressing range, some nonexpressers retained more posttraining state than individual expressers, and atypical turn animals did not form a molecular category.

When retention was averaged by donor line, the estimated difference decreased to 1.1 percentage points in motor cortex (95% CI, −1.0 to 3.3; P = .27) and 0.8 in dorsal striatum (95% CI, −1.2 to 2.8; P = .39). Leave-one-line-out analyses retained a positive point estimate in six of eight iterations for each tissue, but none produced a false-discovery-rate-adjusted result below .05.

No individual methylated region predicted expression or distinguished leftward from rightward behavior. A classifier trained on the selected set did not separate every donor-matched animal and was not considered validated. RNA sequencing identified nineteen genes adjacent to retained regions with nominal group differences at P < .05, including genes related to neuronal development, cytoskeletal regulation, and activity-dependent signaling. None survived correction, the affected genes were inconsistent across donor lines, and genome-wide analysis produced no expression profile uniquely identifying donor-matched animals or turn direction. Routine histology showed no gross cortical, striatal, peripheral nerve, or skeletal muscle abnormality corresponding to the phenotype.

I interpret these results as evidence that incomplete reprogramming can preserve a limited fraction of donor-cell state into later development and that the amount retained may be weakly associated with behavioral expression. I do not interpret them as evidence that DNA methylation stores the acquired action.

Yes, incomplete reprogramming gives us a route by which the condition of the transferred cell can matter after SCNT. It still does not explain why a state measured in a fibroblast should alter the later probability of one historically specific direction or order of movement.

Comparable molecular analysis was not possible in the human cases. Material retained from iPSCs-11 lacked an appropriately matched donor sample, and GIAM-27 tissue was obtained after advanced metabolic deterioration without valid matched donor material. No molecular inference was drawn from either record.

Across the study, the strongest evidence remained behavioral and prospective. The molecular findings provide a candidate developmental context. They do not yet provide the mechanism.

Discussion

This study began with a retrospective observation in one cloned macaque and proceeded to a prospective comparison using paired donor-cell samples collected before and after acquisition of an assigned action. The mouse experiment produced the principal result. Clones derived from posttraining fibroblasts were more likely to develop an additional stable turn during ordinary operant learning, and that turn more often agreed with the direction assigned to the associated donor. They did not enter the chamber knowing the donor task, acquire the reinforcement schedule unusually quickly, or reproduce a complete trained response. Expression also decreased in several animals when the familiar spatial configuration was changed.

I checked each of those possibilities because any one would have changed the interpretation. Spontaneous performance before shaping would have approached conventional memory transfer. Faster acquisition would have supported a more general inherited learning advantage. Equal expression across a substantially altered chamber would have given greater weight to a fixed motor bias. We observed none of those patterns.

The human archive supplied two correspondences of unequal strength, and the molecular study showed that some donor-cell state survived reprogramming without identifying a feature that predicted behavior or specified direction. Taken together, the findings are narrower than memory transfer and stranger than ordinary resemblance. I use behavioral residue for that middle category: a partial, cue-dependent recurrence of an acquired component of donor behavior after nuclear transfer, without recovery of the knowledge, purpose, or recollection that originally made the component useful.

The term is descriptive. I am not using it as a mechanism with a new name.

The prospective mouse comparison carries the greatest inferential weight because pretraining and posttraining cells came from the same donors, leftward and rightward routes were randomly assigned, clones were never exposed to the guide procedure, and behavioral classification was completed before source-cell identity was disclosed. Nuclear genotype was held constant within donor pairs. The assigned direction had no biological relationship to the donor before allocation. Stable inherited preferences for activity, exploration, learning rate, or turning can explain background variation, but they do not by themselves explain why posttraining expression more often agreed with a direction assigned during the donor’s later experience.

The result is also less decisive than the animal count first makes it appear. Donor-matched turns occurred in 7/21 posttraining clones and 1/19 pretraining clones, producing an animal-level P value of .046. At the donor-line level, matching expression occurred in five of eight posttraining lines and one of eight paired pretraining lines, with exact McNemar P = .125.

Yes, one analysis crosses .05 and the other does not. I checked both because twenty-one posttraining clones are not twenty-one independent donor histories. A donor that happens to produce four viable clones should not receive four times the evidentiary weight of a donor that produces one. Clones from the same line share donor genotype, biopsy history, culture, and production conditions. The animal-level analysis describes expression among the organisms produced; the line-level analysis asks whether the effect recurred across independently trained source animals. In this series, there were eight such histories.

I therefore regard the mouse result as evidence of a large but imprecisely estimated effect recurring across several donor lines. I also regard independent replication as necessary. Those positions do not conflict. The animal-level P value is not a certificate that the phenomenon has been established, and the line-level P value is not evidence that nothing occurred. Four lines produced a match only from posttraining cells, one produced matches from both periods, none produced a match only before training, and three produced none in either condition. That distribution is why the result deserves explanation.

The matching pretraining clone is important. It demonstrates directly that donor-concordant direction can arise without a posttraining source cell. Chance agreement, stable laterality, developmental variation, or a donor-specific motor preference are therefore real alternatives. I would be much less interested if all seven posttraining matches came from one unusually productive donor. They did not. Matching expression occurred across five lines, four of which showed no corresponding pretraining match.

The relationship with donor persistence points in the expected direction but does not establish a dose-response effect. Four of five donors with persistent turns produced a matching posttraining clone; one of two donors with intermittent persistence did so; the donor whose turn ceased did not. The sample is eight animals, one persistently turning donor and one intermittently turning donor produced no matching clone, and no line produced matching behavior in every clone. Whatever the source-cell effect was, it behaved probabilistically.

The negative behavioral results help define what that probability affected. Posttraining clones did not reproduce the assigned direction in their first unrewarded exposure, anticipate reward, or know that five presses completed the schedule. They learned the apparatus for themselves. The additional movement emerged during that learning and remained in six animals after the fixed-ratio schedule was replaced by a variable-ratio schedule. The clone adjusted to the new contingency while retaining the redundant return.

That pattern is more consistent with a bias in action organization than with preservation of a fixed response program. Later learning remained necessary. Donor history appears, at most, to have changed which of several available organizations was more likely to stabilize under sufficiently similar circumstances.

The altered-chamber test supports the same boundary. Five of nine stable-turn animals lost the movement or showed it only transiently after lever housing, reward-port placement, and route geometry were changed. Four retained it. Because several spatial features changed together, the experiment cannot identify the controlling cue. I would prefer a cleaner decomposition, and future work should provide one. The present result tells us only that expression was not equally stable across configurations.

The macaque observation fits this interpretation, although it cannot carry the same evidentiary weight. SCNT-M4 reproduced a stable sequence of leftward contact, withdrawal, and second approach that became established in the donor during documented guide misalignment, persisted after repair, and was unnecessary on the clone’s apparatus. The donor chronology remains more informative than the visual resemblance. Two macaques can move an object left or withdraw after failed insertion for many reasons. The interesting feature is that the surviving record shows when the additional movement became useful and that it remained after usefulness disappeared.

The case is still retrospective. Donor and clone were studied within one institution, some staff remained across both periods, and the donor apparatus was unavailable for direct inspection. We found no documented demonstration of the donor sequence to SCNT-M4, no routine use of donor footage during clone training, and no formal route by which the movement was transmitted.

Yes, I checked the equipment records, training records, staff overlap, and access history because institutional transmission is the first explanation I would ask about too. Those records do not contain every conversation that occurred. I can exclude documented transmission. I cannot exclude an undocumented remark later reproduced in training. That is why SCNT-M4 remains the observation that generated the prospective question, not a replication of the mouse experiment.

The human records require greater restraint because they arose from selected clinical archives and did not share one discovery procedure.

iPSCs-11 is the stronger human case. Its lift, full replacement, immediate second lift, and placement against the head were recorded and countersigned before donor provenance was released. Only afterward was the donor found to have acquired the same sequence during an intermittent hook-switch failure, retained it after the instrument was replaced, and performed it before source-cell collection. The order of documentation sharply reduces one form of selection: investigators did not first learn the donor habit and then search the subject record for something resembling it.

It does not eliminate selection altogether. No general human criterion existed before the telephone sequence was noticed, and the observer knew that iPSCs-11 had an SCNT origin. The subject also showed broader repetitive object manipulation. We reviewed that record because perseveration is an obvious alternative. Repetition occurred with other objects, but the same sequence of completing an action, fully resetting the object, repeating it once, and then proceeding was not identified elsewhere. The telephone sequence occurred on five of six contacts. That is why I retain the case. It does not make perseveration disappear.

Exposure is similarly constrained, not eliminated. No corded telephone was identified in the documented residential inventory, no earlier training material contained the double-lift sequence, staff demonstrations used one lift, and the clinical unit was not recorded as having access to donor workplace footage. Direct-care personnel reported no knowledge of the donor habit. Some senior personnel could access both provenance and clinical material, continuous residential audio did not exist, and informal conversations were not archived. The record supports the statement that no documented route of exposure was found. It does not support the stronger statement that exposure was impossible.

The behavioral correspondence also remained procedural. iPSCs-11 did not dial, select numbers, wait for an answer, speak into the receiver appropriately, or demonstrate an understanding of telephone communication. The subject reproduced an ordered redundant component without the knowledge that made the donor’s sequence meaningful.

GIAM-27 is phenomenologically more extensive and causally less constrained. Her pronunciation attracted clinical comment before the present review, and two provenance-blinded reviewers independently described strong German phonological influence and placed the speech within West Central German or Rhine Franconian ranges, principally Palatine and Saarland. After donor provenance was released, both placed GIAM-27 and the donor within the same broad regional grouping. I find that agreement difficult to dismiss.

I also cannot give it the same status as the telephone sequence. Speech depends on anatomy, auditory exposure, social learning, respiratory control, motor development, phonological organization, and experience. GIAM-27 shared the donor’s nuclear genotype and underwent accelerated growth, uneven neurological development, metabolic deterioration, and later respiratory and motor impairment. Detailed biomechanical imaging of the vocal tract was never completed. Several German-like features can arise independently through motor restriction or other linguistic influences.

The auditory history is no cleaner. English was the only language documented in instructional, residential, and clinical programs. No German lesson, intentional accent model, or regular caregiver from the Palatinate was identified, and donor recordings were not recorded as released to the clinical unit. Continuous residential audio did not exist, temporary personnel had incomplete language histories, and incidental media or overheard speech could not be reconstructed. GIAM-27’s question, “What is German?”, is tempting evidence and not very useful. A person can hear a language without knowing its name, and GIAM-27 was not a reliable historian of remote experience.

GIAM-27 therefore remains a boundary observation. The blinded regional agreement deserves reporting. Shared anatomy, abnormal development, incomplete auditory history, archival selection, and chance regional agreement prevent me from treating it as evidence equivalent to the prospective mouse result or the more discrete iPSCs-11 sequence.

The archive cannot estimate prevalence. Six subjects had ever shown sufficient responsiveness for structured assessment, five had adequate comparative records, and iPSCs-11 prompted the review. GIAM-27 was the only additional case retained among four subsequently examined records. Two cases among five records would be a prevalence estimate only if those five formed an unbiased sample with equal survival, neurological organization, cue exposure, and documentation. They plainly do not. Absence of a recorded correspondence is also ambiguous: a residue may have been absent, a relevant cue may never have been presented, or a subject may have lacked the organization required to express it.

I am overexplaining this point because the human numbers are exceptionally easy to misuse. The human records help define candidate manifestations and methodological hazards. They should not be used to infer how common acquired donor effects would be in human clones.

Previous reports of effects following parental olfactory conditioning, early stress, or sperm RNA show that acquired physiological states can influence later development.[4,5] I cite those studies for that limited proposition. Germline inheritance, sperm RNA, parental conditioning, and SCNT are not interchangeable, and none demonstrates transmission of a learned action through a somatic nucleus. They do, however, prevent the objection that experience cannot alter biological material in ways that later matter.

Incomplete reprogramming is consequently an obvious route to examine. In our material, most methylation differences between paired donor fibroblasts were erased in blastocysts, while a small and variable fraction persisted. Donor-matched adult clones showed nominally greater retention of posttraining state in motor cortex and dorsal striatum, but both tissue results had corrected q = .088, the distributions overlapped, and the effects weakened at the donor-line level.

Yes, I think that matters.

No, it does not solve the problem.

The molecular analysis began after behavioral classification and deliberately included every donor-matched animal. Finding an attractive motor-system association under that design is precisely the kind of result that requires replication. No individual methylated region predicted expression, no retained feature distinguished left from right, RNA sequencing produced no corrected signature, and histology showed no corresponding gross abnormality.

I checked direction specifically because a feature that distinguishes posttraining from pretraining is insufficient for the central claim. The behavior was historically directional. A mechanism that cannot account for direction has explained only part of the observation. Epigenetic is an extremely convenient word when an explanation is missing. Naming a route by which donor history could influence development does not explain why that route should preserve the difference between left and right.

The transferred fibroblast nucleus contained neither the donor’s motor cortex nor its striatal circuitry, synaptic weights, practiced sensorimotor sequence, or recollection of training. Any viable account must operate through development. One possibility is developmental convergence: residual donor-cell state might alter the probability with which a developing nervous system forms, selects, or stabilizes one of several available action organizations. Later learning under sufficiently similar cues could then converge preferentially on a pattern resembling the donor’s acquired solution.

I think this is a plausible class of explanation. I do not think it is yet a mechanism. It accommodates a later bias without requiring reconstruction of the donor’s original neural circuit, but it still does not explain specificity to an assigned direction, the order of a redundant telephone sequence, or a regional organization of speech. Historical specificity remains the hardest problem.

Several less exotic explanations also remain for the mouse result. The paired biopsies were separated in time. Donor age, culture history, activity, stress, metabolism, and repetition differed between periods. Handled controls reproduced time, handling, chamber exposure, and reward but not sustained lever pressing, route acquisition, or every physiological consequence of training. A training-associated cellular state might therefore reflect workload or stress independently of learned information. That possibility would weaken a claim about acquired content without necessarily removing a posttraining effect.

SCNT production added variation in oocyte donors, recipient females, manipulation batches, cross-fostering, cages, and postnatal environment. These factors were distributed across conditions where developmental yield allowed, but perfect balance was not possible. Blinded coding reduced expectancy during classification; it could not eliminate every interaction between animals and human experimenters. Random assignment closed one route of bias by preventing investigators from choosing direction according to a donor’s preference, but chance alignment with pre-existing laterality remained possible. The matching pretraining clone demonstrates that directly.

The next experiment should therefore increase independent donor histories before increasing clones per culture. Donors should undergo repeated baseline assessment followed by randomized directional training. Cells should be collected before acquisition, during stable persistence, after extinction, after reversal, and ideally after reacquisition. Cultures from each biopsy should be divided across manipulation batches, oocyte donors, and recipients, with clone rearing and behavioral testing conducted under concealed allocation at a separate facility.

Reversal is the most informative addition. If cells collected after reversal produce clones preferentially expressing the new direction, fixed inherited laterality becomes a much weaker explanation. Persistence of the original direction would instead favor early training, stable motor bias, or a cell-line property. Extinction samples could test whether cellular state tracks overt persistence. The present pretraining/posttraining comparison collapses these hypotheses and cannot distinguish them.

Cue dependence also requires a factorial design. Lever position, reward-port position, route geometry, housing shape, visual texture, odor, and direction of approach should be manipulated separately. Only then can we determine whether expression is linked to a spatial relation, an object configuration, a stage of action, or a more general transition from reward collection back to task engagement.

Molecular intervention should come later. The current study does not identify a prospectively justified target. Editing one nominally associated region now would risk manufacturing a mechanism by selecting it after seeing the phenotype. I would rather repeat the behavior and ask which cellular differences track acquisition, extinction, and reversal across independent lines. Perturbation becomes informative after that.

The human cases do not justify experimental human SCNT replication. They arose from prior clinical programs and are useful only as archival observations. Future human evidence, should relevant records become available, should come from ethically authorized examination of existing archives or care contexts with prospective exposure documentation and independent review.

Several conclusions can therefore be stated clearly. We have not demonstrated transfer of autobiographical memory, identity, language, intention, or knowledge. We have not shown that a fibroblast contains a miniature representation of learned behavior, that every acquired action modifies donor cells in a transmissible way, or that every clone produced from posttraining cells will express a donor-concordant action. Most did not.

I still think the prospective distribution requires explanation. The paired design, randomized direction, recurrence across several donor lines, absence of spontaneous task knowledge, ordinary task acquisition, and dependence on later context together make the result more specific than general behavioral resemblance. Under the conditions examined here, an acquired state of the donor appears capable of altering the probability with which a later clone organizes one component of action.

Whether donor state caused the behavior remains unresolved. How any effect survived nuclear reprogramming remains unresolved. Why a surviving cellular state should preserve the direction, order, or regional structure of a historically acquired action remains the hardest question.

I am aware that this leaves an uncomfortable category between memory and coincidence. At present, that is where I think the data belong.

Methods

Study Design

The study contained four components with different evidentiary status. The macaque component retrospectively investigated an action identified during an existing longitudinal developmental program. The mouse component was designed prospectively after review of that event and compared clones derived from paired fibroblast samples collected from the same donors before and after acquisition of an experimentally assigned directional action. The human component began with documentation of one index archival case before release of donor provenance, followed by a criterion-based review of the remaining eligible records. Molecular analyses began after behavioral classification and were exploratory.

These components were not treated as equivalent repetitions. The prospective mouse comparison carried the prespecified inferential analysis. Macaque and human observations were analyzed as historical cases and were not combined statistically with the mouse data. Molecular analyses examined incomplete donor-state reprogramming and association with behavioral expression; they were not part of the prospective behavioral analysis plan.

Animal work was conducted at a BioDome-funded facility within the Karrillya Conservation Reserve. Human records remained in the BioDome clinical archive and were accessed under a separate authorization. No human nuclear-transfer procedure, clinical intervention, change in care, or behavioral exposure was performed for this study.

For the mouse experiment, donor direction, the stable-turn criterion, test order, the primary donor-line comparison, and planned animal-level secondary comparisons were recorded before clones entered operant shaping. Personnel responsible for clone rearing, testing, video coding, and initial molecular processing did not receive donor direction or source-cell collection period before the relevant classifications were completed.

The number of donor lines was constrained by a predetermined SCNT production series. Transfer attempts were not extended, stopped, or redirected in response to behavioral results. Every clone meeting the prospectively defined health and mobility criteria was assessed, and no eligible animal was removed after source-cell identity was disclosed.

Retrospective Macaque Investigation

The longitudinal cohort included eleven SCNT-derived cynomolgus macaques and fourteen nonclonal controls observed from six through thirty months of age. The program assessed species-typical behavior, object exploration, manual coordination, and acquisition of simple object-directed actions. Nuclear donor recordings were retained as one comparison source among records from unrelated macaques and nonclonal controls.

SCNT-M4 completed a manual insertion task using a cylindrical handled object and a recessed opening in a vertical panel. A trial began when the object was placed within reach and ended after successful insertion, abandonment, or 60 seconds. No shaping procedure required lateral contact, withdrawal after contact, or a second approach. Frontal and overhead cameras recorded each session.

The sequence of interest was defined from SCNT-M4 recordings before the associated donor archive was opened. Reviewers coded movement from first contact between object and opening until insertion or trial termination. The candidate sequence required three ordered components: leftward displacement after contact, withdrawal, and initiation of a second approach. Frequency was calculated from trials in which the object reached the opening and insertion was subsequently completed.

Lateral correction alone was not classified as anomalous. Control animals sometimes displaced or withdrew the object during early acquisition. The candidate sequence had to retain the same order after direct insertion had been acquired and after the correction no longer improved performance.

After definition of the SCNT-M4 sequence, a second reviewer group received relabeled recordings of the associated donor and four unrelated macaques that had completed earlier versions of the task. Reviewers were not told which record belonged to the donor and did not receive the maintenance chronology. They coded all usable trials according to the clone-derived definition. Disagreements were resolved from the complete synchronized trial rather than an isolated clip.

Equipment logs, maintenance entries, training schedules, staff assignments, access records, and surviving pre- and post-service recordings were then reviewed. The original donor apparatus had been removed from service, so its condition could be reconstructed only from documentation and recorded performance. The maintenance entry established the reported fault and repair chronology; it was not treated as direct proof that the fault caused the donor movement.

Personnel records were examined for staff overlap between donor and clone periods. The review could identify formal demonstrations, documented access to footage, and recorded use of donor material, but not every informal conversation. SCNT-M4 was later tested on a second apparatus of the same general dimensions and an altered insertion set differing in object appearance, opening shape, and guide structure. The altered set changed several features at once and tested dependence on the familiar configuration rather than a single cue. Test order was fixed before donor information was released to the animal-care personnel conducting follow-up sessions.

No inferential statistic was applied to the macaque case. Frequencies were reported descriptively, and classification considered chronology, persistence, redundancy, cue dependence, and the available exposure record.

Mouse Donors, Training, and Source-Cell Collection

Twelve adult male mice from an F2 hybrid colony derived from C57BL/6J and DBA/2J founders served as nuclear donors. Eight entered operant training and four served as handled controls. The F2 background permitted genome-wide identity confirmation and retained natural variation in motor and learning traits; the principal comparison nevertheless held nuclear genotype constant through paired samples from each trained donor.

Animals were maintained under a 12-hour light cycle with controlled temperature and humidity, and training occurred during the dark phase. Before chamber exposure, a skin biopsy was obtained under general anesthesia. Biopsy side was balanced across animals. A second biopsy was taken from the opposite side after the training or control interval.

Fibroblasts were isolated by explant culture, expanded under common conditions, and cryopreserved between passages two and four. Paired samples received coded identifiers and were processed in separate culture batches without labels indicating period or condition. Cultures were excluded before nuclear transfer for microbial contamination, abnormal karyotype, failure to enter reversible quiescence, or insufficient viable cells for the predetermined series. No culture was excluded according to later behavior, methylation, or developmental outcome.

Leftward or rightward return was assigned to trained donors by computer-generated permuted blocks within training batch, with four donors per direction. Allocation was completed before acquisition and stored separately from later clone-production codes.

Operant chambers contained a lever, liquid-reward port, and interchangeable internal panels. A computer-controlled pump delivered 20 μl of sucrose solution after the required response. Following magazine training, donors were shaped to press the lever and advanced from continuous reinforcement to a fixed-ratio-five schedule.

During acquisition, a removable guide constrained the path from reward port back to lever, requiring four donors to return around the left side and four around the right. The guide altered the route, not the response requirement. It was removed after each donor completed the fixed-ratio requirement without assistance in three consecutive sessions, and training continued in an unobstructed chamber.

A donor return began when the animal withdrew its head from the reward port and ended at renewed lever contact. Direction was assigned from the path around the longitudinal axis between port and lever. The assigned turn was classified as persistent when it occurred before at least two thirds of completed ratios in each of three consecutive unobstructed sessions. Intermittent persistence and cessation were recorded separately. Posttraining biopsies were obtained only after persistence or cessation had been documented.

Handled controls entered the same type of chamber on schedules yoked to trained donors. Liquid reward was delivered at matched times, the lever was covered, and no guide was installed. They therefore experienced repeated handling, chamber placement, reward consumption, the biopsy interval, and comparable facility procedures without acquiring the lever response or assigned route. This group did not reproduce sustained pressing, route execution, or every physiological consequence of active training; the paired trained-donor samples remained the principal comparison.

Somatic Cell Nuclear Transfer and Rearing

Nuclear transfer used paired pretraining and posttraining fibroblasts from the eight trained donors and second-biopsy cultures from the four handled donors. Metaphase II oocytes were obtained from superovulated B6D2F1 females, enucleated under polarized-light microscopy, and injected with a single quiescent fibroblast nucleus by piezo-assisted injection. Reconstructed oocytes were chemically activated, cultured to the two-cell stage, and transferred to pseudopregnant recipients using established mouse nuclear-transfer methods modified for dermal fibroblasts.[7]

Oocyte donors, manipulation dates, culture batches, and recipient females were distributed across paired conditions wherever developmental yield permitted. Embryos from paired samples of the same donor were not knowingly transferred to the same recipient. Nuclear-transfer and allocation personnel received coded cultures without donor training information.

Production followed a predetermined schedule. A series was not repeated because one condition produced fewer live births, and production was not expanded after a concordant clone appeared. Live-born animals were cross-fostered when required. After weaning, clones were housed in mixed-source groups as far as sex, age, health, and yield allowed. No operant chamber, guide panel, or donor recording was present in rearing rooms.

Eligibility required survival to the scheduled testing age, absence of a veterinary condition requiring removal, and free movement through the apparatus. Nineteen eligible clones came from pretraining samples, twenty-one from posttraining samples, and twelve from handled-control second samples. These were all eligible animals produced in the predetermined series.

Blinding and Behavioral Assessment of Clones

Source cultures, embryos, clones, recordings, and molecular samples received linked coded identifiers. Rearing and testing personnel knew husbandry identity but not donor direction, collection period, or handled-control status. Video coders received session and animal codes only. The stable-turn criterion, unscorable-trial rules, and initial classifications were finalized before donor direction and collection-period codes were released. Donor-line identity was released afterward for clustered and paired analyses.

At the start of testing, each clone received a 20-minute unrewarded exposure to the unobstructed chamber. Lever and reward port were present, but no reward was delivered. This tested for spontaneous directional return, repeated lever sequence, or organized donor-like performance before shaping.

Animals then underwent magazine training, lever shaping, and progression to fixed-ratio five using the donor protocol except that guide panels were never installed. Overhead and lateral cameras recorded activity. Automated tracking supplied position and orientation estimates, but final turn classification used the complete movement because climbing, rearing, grooming, and partial port entry could distort automated paths.

A return began when the animal withdrew from the reward port and ended at the next lever contact. Trials with prolonged grooming, climbing, staff intervention, failure to return, or an obstructed view remained in the session record but were not assigned a direction; no missing direction was imputed.

Before unblinding, a stable turn was defined as the same direction before at least two thirds of completed ratios in each of three consecutive sessions. A turn was donor-matched only when it agreed with the direction randomly assigned to the associated trained donor. For pretraining clones, matching was defined against the direction assigned after the pretraining biopsy, thereby controlling donor genotype and pre-existing characteristics while remaining historically prior to training. Opposite and inconsistent stable movements were recorded separately. Handled-control clones could develop a stable turn but could not be classified as donor-matched.

A stratified reliability set included every trial from animals classified with a stable movement and randomly selected sessions from animals without one. Two reviewers independently coded complete trials without group or donor-direction information. Disagreements were resolved before code release. When automated and manual classifications differed, complete-trial manual review prevailed.

Sessions to stable fixed-ratio-five performance, reward yield, incorrect reward-port approaches, completed ratios, omitted trials, lever contacts, and session-by-session acquisition were retained independently of turn classification. These measures tested whether the additional movement accompanied a general difference in learning or reward performance.

After stable classification, the fixed-ratio schedule was replaced with a variable-ratio schedule having the same mean response requirement. This removed the constant grouping of five presses while preserving responding and reward. The nine posttraining clones with stable turns were then tested in a second chamber with a different lever housing and a reward-port position that did not reproduce the familiar return path. The manipulation assessed configuration dependence, not a single cue. Neither postclassification test could alter the original classification.

Human Archive and iPSCs-11 Index Case

The human review was restricted to records from prior clinical SCNT programs. Registry identifiers were assigned when a developmental program, source-cell line, or clinical attempt entered its archive and were retained after failed development, fetal loss, early death, or survival without sustained responsiveness. Registry numbers therefore did not count live-born or behaviorally assessable individuals. The iPSCs prefix in iPSCs-11 was a legacy registry label and did not denote induced pluripotent stem cells.

Six archived subjects had at some point shown sufficient repeatable responsiveness for structured assessment. Five had longitudinal audiovisual material, clinical documentation, and donor provenance adequate for comparison, including iPSCs-11 and GIAM-27. Eligibility meant that observable behavior and adequate records existed; it was not treated as a retrospective standardized diagnosis of consciousness.

iPSCs-11 was not found through a blinded search of all five records. During routine facility work, D.S.E.L. observed an object-familiarization session from an adjacent monitoring area. He knew the subject had an SCNT origin but did not know the donor’s identity, occupation, workplace history, or telephone behavior.

The complete session recording was preserved before a donor file was requested. A description was entered in the institutional record and divided into initial lift, complete replacement on the cradle, immediate second lift, and placement against the side of the head. An archive officer countersigned the entry before donor access was authorized. Two additional reviewers without donor access confirmed the component order from complete interactions rather than selected clips.

The disconnected telephone was inspected. Receiver, cradle, hook switch, and cord moved normally; one lift was sufficient, and replacement followed by a second lift had no mechanical effect. Before first contact, the examiner demonstrated one lift, brief placement beside the head, and one replacement. Ear laterality was not treated as independent evidence because it could reflect mirrored imitation, posture, handedness, or shared biological laterality.

All surviving telephone contacts were reviewed from first presentation. Receiver, cradle, hand, and head positions were coded separately, and the wider object-handling archive was examined for repetition, perseveration, single repetitions after reset, and staff-demonstrated action.

Only after the sequence was entered and countersigned was the donor file released. Every usable donor telephone interaction was reviewed chronologically in relation to the reported equipment fault and replacement. Workplace maintenance records independently established intermittent hook-switch failure and the reported effect of reseating the receiver; they did not describe the donor movement and were not treated as proof of acquisition. Source-cell collection was then placed in the chronology to determine whether donor acquisition and post-repair persistence preceded biopsy.

Exposure review covered residential inventories, prior object sessions, staff demonstrations, training material, accessible media, archive-access records, personnel assignments, and direct-care interviews. The review distinguished absence of a documented route from proof that no exposure occurred. Continuous residential audio had not been maintained, and informal communication could not be reconstructed completely.

No inferential statistic was applied. Classification considered documentation order, recurrence, sequence, redundancy, donor chronology, source-cell timing, exposure record, and alternatives.

Subsequent Human Review and GIAM-27

No general screening criterion existed before iPSCs-11. After the index sequence was documented and before the remaining records were compared with donor provenance, eligibility and reporting criteria were fixed.

The remaining four records were first reviewed without donor histories. Reviewers coded repeated object-directed, oral-motor, articulatory, and other procedural sequences. A candidate had to occur on more than one unprompted occasion, be distinguishable from staff demonstration, and contain an element unnecessary for the immediate task. Common postural adjustments, isolated movements, direct same-session imitation, and actions that could not be placed reliably in time were not advanced.

Donor provenance was released only after a candidate description had been entered in a dated record. Complete donor material was then compared where available, and residential inventories, staff demonstrations, training records, media access, personnel assignments, and clinical notes were reviewed for transmission routes. A case remained reportable when the subject-side feature had been defined before comparison, the donor file contained a sufficiently specific correspondence, and no documented direct model was identified. Three of the four records produced no reportable correspondence. GIAM-27 was retained as the only additional boundary case.

The GIAM-27 archive contained eleven audiovisual sessions across fourteen weeks: seven structured developmental assessments and four routine clinical interviews. Approximately forty-three minutes of intelligible elicited or spontaneous English speech were available.

Two reviewers experienced in German regional phonetics received the complete usable set under coded identifiers without the subject’s name, SCNT history, donor identity, donor origin, or study hypothesis. They described pronunciation, assessed possible influence from another language, and supplied regional alternatives only where supported. Initial classifications were signed before donor provenance was released.

Review considered recurrent segmental and prosodic features across words and sessions, including interdental fricatives, initial English /w/, word-final obstruents, rhotics, vowel placement, phrase timing, and pitch movement. Lexical transcripts were kept separate from narrow phonetic transcriptions so that English content was not mistaken for German vocabulary or grammar.

After initial classifications were locked, reviewers received coded donor English samples mixed with unrelated German speakers and ranked regional compatibility where possible. They were not asked to infer identity from pitch or voice quality.

Exposure review covered instructional material, clinical records, retained media, residential-language documentation, donor-archive access logs, staff rosters, caregiver interviews, and available language histories. Continuous residential audio did not exist, and language histories were incomplete for some temporary personnel. The review also considered shared genotype, accelerated growth, respiratory instability, neurological development, motor decline, and absence of detailed vocal-tract imaging. No inferential probability or archive-wide frequency estimate was calculated.

Molecular Samples and Analyses

Frozen paired fibroblast aliquots were available from all eight trained donors, and time-separated samples from all four handled controls. Additional material included untransferred SCNT blastocysts and terminal motor cortex, dorsal striatum, cerebellum, and quadriceps from twenty-four behaviorally classified clones collected within a common age range under coded identifiers.

The adult set was selected after behavioral classification. It included all twenty-one posttraining clones: seven donor-matched, one opposite, one alternating, and twelve without a stable turn. Three pretraining clones were added as source-period references: the two with stable turns and one without. The handled-control clone with a stable turn was not included because principal donor-state regions were defined from paired trained-donor samples and the handled donor had no assigned direction.

The principal adult expression comparison was restricted to posttraining clones and contrasted the seven donor-matched animals with the twelve posttraining animals without a stable turn. Opposite and alternating animals were displayed separately. The pretraining references were used to inspect source-period behavior of the retention metric but not in the principal expression test. This restriction was specified before the final molecular comparison.

Genome-wide single-nucleotide-polymorphism genotyping confirmed nuclear identity between each clone tissue and its corresponding donor culture. Samples with unresolved mismatch, interchange, or inadequate coverage were to be excluded before epigenetic analysis. Mitochondrial haplotypes were compared with oocyte-donor records, and genotyping data were examined for large acquired copy-number changes. Identity and quality-control decisions were completed without behavioral interpretation, and no sample was removed because its result failed to support the phenotype.

DNA methylation was measured by reduced-representation bisulfite sequencing.[8] Libraries were prepared in randomized batches containing pretraining, posttraining, handled-control, blastocyst, and adult samples. Conversion efficiency, mapped-read minimums, duplication limits, and coverage requirements were established before behavioral codes were released.

Differentially methylated regions were identified separately within each paired pretraining and posttraining donor. A qualifying region required multiple covered CpGs, sufficient depth in both samples, an absolute regional difference above the predetermined threshold, and consistent direction across covered sites. Equivalent time-separated comparisons were performed in handled donors. Regions were defined within donor pairs rather than pooled because no universal posttraining signature was assumed.

A donor-state region was considered retained in a blastocyst or adult tissue when adjusted methylation remained shifted in the source-state direction beyond the predetermined threshold. Adjustment accounted for sequencing batch and tissue class where applicable. Retention was summarized as the proportion of qualifying source-associated regions remaining shifted in that direction. No feature was classified as encoding direction unless it distinguished left-trained from right-trained lines independently of general posttraining status.

RNA sequencing was performed in motor cortex and dorsal striatum from the same adult set. Initial analysis was restricted to genes within or adjacent to retained methylation regions, followed by genome-wide analysis. Models accounted for sequencing batch and donor line where estimable. Candidate-region and genome-wide false-discovery rates were controlled separately. Nominal genes were retained descriptively but not treated as confirmed signatures when corrected thresholds were not met.

Routine histology examined cortical lamination, striatal organization, peripheral nerve, and skeletal muscle under coded identifiers. It was intended to detect gross anatomical or degenerative explanations and could not exclude circuit-level developmental differences.

Comparable human molecular analysis was not attempted. iPSCs-11 lacked matched donor material of the same tissue and collection condition. GIAM-27 material was affected by advanced metabolic disease, and no adequate matched donor sample was available.

Statistical Analysis

The prespecified prospective outcome was a stable turn matching the direction assigned to the associated trained donor.

Because multiple clones could originate from one donor and culture, the primary confirmatory comparison was performed at the donor-line level. A line was positive within a collection period when at least one eligible clone developed a stable donor-matched turn. Paired pretraining and posttraining outcomes were compared with a two-sided exact McNemar test. This definition prevented a donor producing several viable clones from receiving proportionally greater evidentiary weight.

Animal-level comparisons were prespecified as secondary. Donor-matched proportions in posttraining and pretraining clones were compared with a two-sided Fisher exact test. Risk difference was reported with a 95% Newcombe score interval and an unadjusted odds ratio. Development of any stable turn, irrespective of direction, was compared among posttraining, pretraining, and handled-control clones using pairwise two-sided Fisher exact tests. Handled controls were excluded from donor-matched comparisons because no direction had been assigned.

Sessions to stable fixed-ratio-five performance were analyzed with a discrete-time mixed-effects time-to-criterion model containing source-cell group and donor identity, with cage added where stable estimation permitted. Reward yield used a repeated-measures mixed model containing group, session, and their interaction, with animal nested within donor line. Incorrect reward-port approaches used a count model with the same fixed effects and grouping structure. Residual and dispersion diagnostics were examined; when a more complex grouping structure did not converge, the prespecified reduced model retained donor line and repeated animal observations.

Variable-ratio and altered-chamber outcomes were reported descriptively because only the nine posttraining stable-turn clones entered those tests. No inferential test was applied to the macaque event, iPSCs-11, GIAM-27, or the number of human cases.

For adult methylation, the principal animal-level comparison contrasted the seven posttraining donor-matched clones with the twelve posttraining clones without a stable turn. Tissue-specific retention scores were compared by two-sided rank-based tests, with the four tissues corrected together using Benjamini–Hochberg. Donor-line sensitivity analysis used a model containing donor line and expression followed by cluster resampling at the donor-line level and leave-one-line-out repetition. Opposite and alternating movements were not combined with donor-matched expression.

RNA-sequencing false-discovery rates were controlled by Benjamini–Hochberg, with candidate genes and genome-wide analysis treated as separate prespecified families. No classifier was considered validated without evaluation outside the selected twenty-four-clone set.

No formal power target determined donor-line number. The study lacked a prior donor-line estimate and was constrained by the predetermined feasible SCNT series. The result should therefore be read through effect estimates, donor-line distribution, and uncertainty rather than a binary threshold alone.

All tests were two-sided. No behavioral or molecular value was imputed. Exact probabilities were reported for sparse categorical comparisons, and effect estimates received confidence intervals where applicable. Analyses were performed in R version 3.5. Scripts and processed data were retained with the supplementary materials.

Ethics Statement

Animal Research

Procedures undertaken specifically for the prospective mouse experiment were reviewed and authorized by the BioDome Animal Use Review Committee and attending veterinary director. Authorization covered donor biopsies, operant training, somatic cell nuclear transfer, embryo transfer, clone rearing, behavioral assessment, terminal tissue collection, and prospectively defined veterinary removal criteria.

Health assessment and removal decisions were made without source-cell period or donor-direction information. No animal remained in testing after meeting a veterinary removal criterion, and no eligible animal was removed because its behavior failed to support the hypothesis.

The macaque component began as retrospective examination of records generated during an existing developmental program. Subsequent testing of SCNT-M4 with replacement apparatuses was authorized under an amendment permitting non-invasive behavioral assessment. The amendment covered familiar and altered insertion sets and did not permit deprivation, invasive intervention, or recreation of the donor apparatus fault.

Human Archival Research

The human component was restricted to retrospective analysis of existing clinical and developmental records. No human nuclear-transfer procedure, clinical intervention, modification of care, language exposure, object exposure, or additional behavioral assessment was performed for this study.

Access to coded records was authorized by the BioDome Clinical Records Review Panel. The panel waived additional consent under the program agreements and custodial provisions governing the archive. Identifying client information was withheld from investigators responsible for initial behavioral and phonetic classifications, and donor provenance was released only through the staged procedures described above.

The authorization applied to secondary use of existing records. It did not constitute independent ethical review of the originating human SCNT programs, retrospectively authorize procedures performed within them, or establish that those programs had received equivalent review.

This distinction is important. Authorization to examine an archive is not ethical validation of the events that created it.

Complete human recordings remained within the controlled clinical archive. Investigators could review only material required for the authorized comparison and had no independent authority to copy, redistribute, or release restricted records.

Data Availability

Trial-level mouse behavioral data, donor-line assignments, source-cell period codes, stable-turn classifications, unscorable-trial flags, task-acquisition measures, and analysis-ready tables are provided in Supplementary Data 1 and 2. The allocation table is included after replacement of operational production identifiers.

Processed methylation-region definitions, donor-state retention measurements, tissue summaries, and expression results are provided in Supplementary Data 3. R scripts for behavioral, methylation, expression, sensitivity, and leave-one-line-out analyses are provided in Supplementary Data 4.

The released materials preserve the distinction between individual clones and independent donor lines. Both animal-level and line-level tables are supplied so that the result is not reproducible only under one choice of experimental unit. Information identifying oocyte donors, recipient females, proprietary fibroblast preparations, manipulation personnel, or protected production batches has been replaced with study codes.

Raw sequence files, complete nuclear-transfer production records, and the key linking public study codes to restricted operational identifiers are maintained in the BioDome Controlled Research Archive. Access requires a scientifically specified request, institutional data-use agreement, and technical and confidentiality review by the archive custodian. BioDome retains authority over proprietary nuclear-transfer records; the corresponding author cannot independently waive those restrictions.

Complete macaque recordings, human audiovisual material, donor provenance files, registry records, residential inventories, personnel interviews, and access logs cannot be deposited publicly because they contain biometric, occupational, medical, and historical information that cannot be removed reliably without also removing information necessary to evaluate chronology, exposure, and behavioral correspondence.

Redacted chronologies and classification evidence for iPSCs-11 and GIAM-27 are provided in Appendix A and Appendix B. They do not reproduce the complete underlying records. Access to restricted human material requires separate authorization from the Clinical Records Review Panel and is not granted automatically with access to the mouse data.

Failure to obtain restricted records should not be interpreted as independent confirmation of the case classifications. The public appendices permit examination of the reported chronology and reasoning. They do not allow an outside investigator to reproduce every archival judgment from the original audiovisual and provenance material.

Funding

This study received full financial, material, and infrastructural support from BioDome. The company provided source-cell cultures, nuclear-transfer facilities, animal housing, the clinical archive, sequencing resources, computing infrastructure, and salaried or contracted personnel. No external grant supported the work.

BioDome determined which archival materials could be accessed under existing client agreements and reviewed the manuscript for protected client information, facility security, and proprietary technical content. That review affected the level of procedural, operational, and identifying detail disclosed. It did not alter the prospectively defined mouse criteria, coded outcomes, donor-line classifications, statistical results, or the distinction between prospective and retrospective evidence.

The authors nevertheless worked within an archive and production system owned and controlled by the funder. That institutional dependence cannot be removed by stating that the final analysis was unchanged.

Competing Interests

All authors are BioDome employees, contracted investigators working within BioDome programs, or personnel whose participation was supported through BioDome facilities.

BioDome owns or controls the donor-cell lines, SCNT production records, animal-development records, clinical archives, restricted audiovisual material, and associated technical intellectual property examined in this study. The company may obtain commercial, legal, reputational, or strategic benefit from research concerning developmental consequences of nuclear reprogramming, including possible persistence of donor-cell state.

The authors had no independent authority to release restricted program records outside the conditions described in Data Availability. D.S.E.L. had responsibility for the final scientific interpretation and manuscript text but could not compel release of proprietary or protected source material.

These interests do not determine whether the reported behavioral distribution occurred. They do affect which underlying records can be inspected independently and therefore form part of the study’s evidentiary limitation.

Acknowledgments

We thank the animal-care and veterinary personnel at the Karrillya facility for maintaining longitudinal health and behavioral records and applying removal criteria without source-cell information. We thank the nuclear-transfer and nursery personnel who maintained coded production and rearing procedures despite unequal developmental yield across donor lines.

We also thank the archive technicians who preserved apparatus logs, workplace recordings, clinical records, donor provenance files, access histories, and legacy registry documentation. The historical analyses would not have been possible from selected behavioral clips alone.

We thank the reviewers who completed blinded macaque movement comparisons and phonetic classifications, including those who recorded disagreement and regional uncertainty rather than forcing greater precision than the material supported. We further thank the personnel who prepared redacted chronologies while preserving the order in which subject observations and donor information were released.

Participation in animal care, archive preservation, clinical review, phonetic classification, technical review, or manuscript clearance does not imply agreement with the interpretation of behavioral residue proposed here.

Author Contributions

D.S.E.L. conceived the study, initiated investigation of the SCNT-M4 observation, and developed the behavioral-residue framework. D.S.E.L. and E.P.S. designed the prospective mouse comparison. D.S.E.L. documented the iPSCs-11 index observation before donor provenance was released, directed the subsequent human review, determined the evidentiary treatment of the human cases, and wrote the original manuscript.

E.P.S. developed donor-training and clone-testing procedures, supervised operant training, behavioral assessment, and blinded video coding, maintained the mouse behavioral dataset, and participated in review of macaque and human action sequences.

N.F. performed genotyping, methylation, and expression analyses; developed statistical and bioinformatic workflows; conducted donor-line sensitivity analyses; and maintained the molecular and analysis-script archives.

A.M. supervised source-cell preparation, fibroblast culture, somatic cell nuclear transfer, embryo culture, and allocation of reconstructed embryos, and maintained restricted records linking donor biopsies, coded cultures, oocyte donors, recipients, and clone production.

L.v.T. provided veterinary oversight, established health-related removal criteria, supervised follow-up assessment of SCNT-M4, and contributed to tissue collection and histological interpretation.

G.K.P. established eligibility for the human archival review, examined the clinical and developmental histories of iPSCs-11 and GIAM-27, reviewed exposure documentation, and advised on medical, developmental, and anatomical alternatives.

M.V. obtained institutional funding and access to BioDome facilities, authorized staff and archival resources, and supervised the broader research program. M.V. did not perform blinded behavioral or phonetic classifications.

D.S.E.L., E.P.S., and N.F. performed the integrated analysis. D.S.E.L. made final decisions concerning evidentiary hierarchy and interpretation. All authors reviewed the manuscript, approved submission, and accepted responsibility for the accuracy of their contributions.